KR102602329B1 - Antibodies specific for CD3 and their uses - Google Patents

Abstract

The present invention provides a new antibody that specifically binds to CD3 and its use. Additionally, the present invention provides methods of treatment and diagnosis using the antibodies and antigen-binding fragments provided herein.

Description

Antibodies specific for CD3 and their uses

Sequence Listing

This application was filed electronically via EFS-Web and includes a sequence listing in .txt format submitted electronically. The .txt file was created on May 13, 2019 and contains a sequence listing titled “PC72375-PRV2_SequenceListing_ST25_05132019.txt” with a size of 100 KB. The sequence listing included in the .txt file is part of this application and is incorporated by reference in its entirety.

Technology field

The present invention relates to antibodies and antigen-binding fragments thereof that specifically bind to CD3, and methods of using the same. The present invention also relates to multispecific antibodies that bind CD3 and target antigens, and methods of using the same.

Cluster of differentiation 3 (CD3) is a homodimeric or heterodimeric antigen expressed on T cells in association with the T cell receptor complex (TCR) and is required for T cell activation. T cell activation is a complex phenomenon dependent on the participation of various cell surface molecules expressed on the responding T cell population. Functional CD3 is formed from the dimeric association of two of the four chains (epsilon, zeta, delta and gamma). For example, the antigen-specific T cell receptor (TCR) contains two clonally distributed integral membrane glycoprotein chains (alpha and beta (α and β)) that are non-covalently associated with a low molecular weight variant protein complex commonly designated CD3. or gamma and delta (γ and δ)). Additionally, CD3 dimer configurations include gamma/epsilon, delta/epsilon, and zeta/zeta.

Antibodies against CD3 have been shown to cluster CD3 on T cells, resulting in T cell activation in a manner similar to binding of the TCR by peptide-loaded MHC molecules. Therefore, CD3 antibodies may be useful for therapeutic purposes related to T cell activation.

The present invention provides antibodies that bind to CD3, and compositions, such as pharmaceutical compositions, comprising one or more CD3 antibodies. Antibodies according to this aspect of the invention are particularly useful for targeting T cells expressing CD3 and stimulating T cell activation, for example under circumstances where T cell mediated killing is beneficial or desirable. CD3 antibodies of the invention may be included as part of a multispecific antibody, e.g., a bispecific antibody, that induces CD3-mediated T cell activation against specific cell types, such as tumor cells or infectious agents.

In one aspect, the invention provides an antibody that specifically binds to CD3, wherein the antibody comprises: (a) a variable heavy chain (VH) shown in SEQ ID NO: 2, 4, 5, 7, 10, 12 or 35; ) VH region comprising VH complementation-determining region 1 (VH CDR1), VH complementation-determining region 2 (VH CDR2), and VH complementation-determining region 3 (VH CDR3) of the sequence; and/or (b) VL complementary determining region 1 (VL CDR1), VL complementary determining region 2 ( VL region, including VL CDR2) and VL complement determining region 3 (VL CDR3).

In one embodiment of this aspect, the CD3 antibody comprises: (a) (i) a VH CDR1 comprising the sequence of SEQ ID NO: 13, 14 or 15; (ii) a VH CDR2 comprising the sequence of SEQ ID NO: 16, 17, 19, 20, 21, 22, 23 or 24; and (iii) a VH region comprising a VH CDR3 comprising the sequence of SEQ ID NO: 18 or 25; and/or (b) (i) SEQ ID NO: VL CDR1 comprising the sequence of SEQ ID NO: 26, 29, 30, 32, 91 or 92; (ii) VL CDR2 comprising the sequence of SEQ ID NO: 27 or 31; and (iii) a VL region comprising a VL CDR3 comprising the sequence of SEQ ID NO: 28 or 33.

In some such embodiments, the invention provides an antibody wherein (a) the VH region comprises the sequence of SEQ ID NO: 2, 4, 5, 7, 10, 12 or 35; (b) The VL region includes the sequence of SEQ ID NO: 1, 3, 6, 8, 9, 11, 34, 87 or 89.

In other embodiments, the antibody is SEQ ID NO: 1 and 2; SEQ ID NO: 3 and 2; SEQ ID NO: 3 and 4; SEQ ID NO: 3 and 5; SEQ ID NOs: 6 and 7; SEQ ID NOs: 8 and 7; SEQ ID NOs: 6 and 4; SEQ ID NOs: 8 and 4; SEQ ID NOs: 9 and 10; SEQ ID NOs: 9 and 7; SEQ ID NO: 11 and 12; SEQ ID NOs: 34 and 35; SEQ ID NOs: 9 and 4; SEQ ID NOs: 87 and 4; and a VL/VH amino acid sequence pair selected from the group consisting of SEQ ID NOs: 89 and 4.

In some embodiments of each of the preceding, the antibody is a Fab, Fab fragment, F(ab) ₂ fragment, Fv fragment, single chain Fv fragment, disulfide stabilized Fv fragment, single chain antibody, monoclonal antibody, chimeric antibody, double antibody. Specific antibodies, trispecific antibodies, multispecific antibodies, bispecific heterodimeric diabodies, bispecific heterodimeric IgG, polyclonal antibodies, labeled antibodies, humanized antibodies, human antibodies and fragments thereof is selected from the group consisting of

In specific embodiments, the antibody is a bispecific antibody. In some such embodiments, the bispecific antibody specifically binds to a tumor antigen.

In some embodiments, the antibody of the invention further comprises a human VH framework or a humanized VH framework, and a human VL framework or a humanized VL framework. In some such embodiments, the antibody is a humanized antibody.

The present invention further provides a pharmaceutical composition comprising the CD3 antibody of the invention disclosed herein and a pharmaceutically acceptable carrier.

In another aspect, the present invention provides a method of modulating a T cell-mediated immune response in an individual in need thereof, comprising administering to the individual an effective amount of an antibody or pharmaceutical composition disclosed herein. This includes regulating the immune response in the subject.

In one aspect, the invention provides a method of inhibiting the growth of tumor cells in an individual, comprising administering to said individual a bispecific antibody, trispecific antibody, multispecific antibody, bispecific heterodimer, or other antibody disclosed herein. and administering a diabody or bispecific heterodimeric IgG in an amount effective to inhibit the growth of tumor cells.

In some such embodiments, the tumor cells are cancerous. In specific embodiments, the cancer is selected from the group consisting of breast cancer, ovarian cancer, thyroid cancer, prostate cancer, uterine cancer, lung cancer, bladder cancer, endometrial cancer, head and neck cancer, testicular cancer, glioblastoma, and cancer of the digestive system.

In one embodiment, the cancer of the digestive system is selected from the group consisting of esophageal cancer, stomach cancer, small intestine cancer, colon cancer, rectal cancer, anal cancer, liver cancer, gallbladder cancer, appendix cancer, bile duct cancer, and pancreatic cancer.

In one embodiment, the invention provides an antibody, or a pharmaceutical composition comprising the antibody, for use in therapy.

In another embodiment, the invention provides an antibody of the invention in the manufacture of a medicament for use in therapy. In some such embodiments, treatment includes activation of a cytolytic T cell response.

In one aspect, the invention provides a polynucleotide comprising a nucleotide sequence encoding an antibody disclosed herein. In another aspect, the invention provides vectors containing such polynucleotides.

In another aspect, the invention provides host cells comprising the vectors disclosed herein. In some such embodiments, the host cell recombinantly produces an antibody disclosed herein. In specific embodiments, the host cell is selected from the group consisting of bacterial cell lines, mammalian cell lines, insect cell lines, and yeast cell lines. In certain embodiments, the mammalian cell line is a CHO cell line.

In one aspect, the invention provides a method of producing an antibody disclosed herein, comprising culturing a host cell under conditions that result in production of the antibody disclosed herein, and purifying the antibody from the culture supernatant.

In another aspect, the invention provides use of a CD3 antibody, polynucleotide, vector, or host cell disclosed herein in the manufacture of a medicament for the treatment of a disorder.

In one aspect, the present invention provides an antibody or pharmaceutical composition for use in a method of modulating a T cell-mediated immune response in an individual in need of such modulation. In specific embodiments, the invention provides antibodies or pharmaceutical compositions for use in a method of inhibiting the growth of tumor cells in an individual.

In another aspect, the present invention provides an antibody or pharmaceutical composition for use in the treatment of cancer, optionally comprising breast cancer, ovarian cancer, thyroid cancer, prostate cancer, uterine cancer, lung cancer, bladder cancer, endometrial cancer, head and neck cancer, It is selected from the group consisting of testicular cancer, glioblastoma and cancer of the digestive system.

In another aspect, the invention provides an antibody or pharmaceutical composition for use in the treatment of cancer, wherein a T cell mediated immune response is modulated or the growth of tumor cells is inhibited.

Other embodiments will become apparent from review of the detailed description. When an aspect or embodiment of the invention is described in terms of a Markush group or other alternative group classification, the invention covers the entire group listed as a whole, as well as each member of the group, all possible subgroups of the main group, and any member of the group. Includes major groups in which one or more are absent. Additionally, the present invention contemplates the express exclusion of one or more members of any group from the claimed invention.

Figure 1 shows a heterodimer-promoting domain having a first heterodimer-promoting domain and a second heterodimer-promoting domain, comprising an Fc chain optimized to associate via “knob-in-hole” association. A schematic diagram of the structure of a bispecific antibody is provided.
Figure 2 shows the results of a differential scanning calorimetry thermogram showing the heat capacity of GUCY2c-H2B4 and GUCY2c-2B5 bispecific antibodies as a function of temperature.
Figure 3 depicts the binding of GUCY2c-H2B4 (GUCY2c-0247) and GUCY2c-2B5 (GUCY2c-0250) bispecific antibodies measured to naive human T cells using flow cytometry.
Figure 4 depicts the results of in vitro cytotoxicity mediated by GUCY2c-H2B4 (GUCY2c-0247) and GUCY2c-2B5 (GUCY2c-0250) bispecific antibodies, using cell viability data.
Figure 5 shows the potential energy surface (PES) of the H2B4 Fv region from the x-ray crystal structure. It includes three views highlighting the VH region, VH/VL interface, and VL region. CDRs with excessive positive charge are marked in the figure for reference. Dark black represents positive charge, and white represents negative charge.
Figure 6 shows the spatial aggregation propensity surface (SAP) of the Fv region of H2B4. Regions with enriched hydrophobic residues were labeled.
Figure 7 shows the heterogeneity of bispecific antibodies containing CD3 antibody binding domains H2B4, 2B5 and 2B5v6, using capillary gel electropherograms.
Figure 8 depicts the heat capacity of the GUCY2c-1608 (GUCY2c-2B5v6) bispecific antibody as a function of temperature, using differential scanning calorimetry (DSC) thermometry.
Figure 9 depicts GUCY2c-1608 (GUCY2c-2B5v6) bispecific antibody binding to naive T cells.
Figure 10 depicts cell viability data for in vitro cytotoxicity mediated by the GUCY2c-1608 (GUCY2c-2B5v6) bispecific antibody.
Figures 11A-11F depict in vitro cytokine release measurements using the GUCY2c-1608 (GUCY2c-2B5v6) bispecific antibody. The bispecific antibody recruits naïve human T cells to GUCY2c-expressing T84 cells and induces cytokine release in vitro. Luminex-based assays upregulated human IFN-gamma (Figure 11A), IL10 (Figure 11B), IL2 (Figure 11C), IL4 (Figure 11D), IL6 (Figure 11E), and TNF-alpha (Figure 11F). showed control.
Figure 12 depicts dose-dependent tumor growth inhibition by GUCY2c-1608 (GUCY2c-2B5v6) bispecific antibody in colorectal cancer patient derived xenograft PDX-CRX-11201 in an adoptive transfer system.
Figure 13 depicts dose-dependent tumor growth inhibition of GUCY2C-1608 (GUCY2c-2B5v6) bispecific antibody in colon cancer cell line xenograft LS1034 in an adoptive transfer system.
Figure 14A shows the results of cell viability data showing in vitro cytotoxicity mediated by the FLT3-2B5v6 bispecific antibody at different effector to target (E:T) ratios using Flt3 expressing MV4-11 cells. Figure 14B shows the results of cell viability data showing in vitro cytotoxicity mediated by FLT3-2B5v6 bispecific antibody at different effector to target (E:T) ratios using Flt-3 expressing EOL-1 cells. .
Figure 15 depicts binding of the low affinity 2B5v6 variant to Jurkat cells expressing CD3.
Figure 16 depicts a graph summarizing the results of a study demonstrating anti-GUCY2C bispecific T cell mediated cytotoxicity using anti-CD3 variants.

The invention disclosed herein provides antibodies that specifically bind to CD3 (e.g., human CD3), including full-length antibodies or antigen-binding fragments thereof. Additionally, the present invention provides polynucleotides encoding the antibodies, compositions containing the antibodies, and methods of producing and using the antibodies. Additionally, the present invention provides methods of treating disorders using the antibodies described herein.

general skills

The practice of the present invention, unless otherwise indicated, will employ conventional techniques in molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the scope of the present invention. These techniques are described in literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; [Oligonucleotide Synthesis (M.J. Gait, ed., 1984)]; [Methods in Molecular Biology, Humana Press]; [Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press]; [Animal Cell Culture (R.I. Freshney, ed., 1987)]; [Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press]; [Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-1998) J. Wiley and Sons]; [Methods in Enzymology (Academic Press, Inc.)]; [Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.)]; [Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987)]; [Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987)]; [PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994)]; [Current Protocols in Immunology (J.E. Coligan et al., eds., 1991)]; [Short Protocols in Molecular Biology (Wiley and Sons, 1999)]; [Immunobiology (C.A. Janeway and P. Travers, 1997)]; [Antibodies (P. Finch, 1997)]; [Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989)]; [Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000)]; Fully explained in [Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999)]; [The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995)] In some instances, terms with commonly understood meanings are defined herein for clarity and/or convenient reference, and the inclusion of such definitions herein does not necessarily constitute a substantive difference to the commonly understood meaning in the art. It should not be interpreted as necessarily indicating.

The invention will be described in detail by reference using the following definitions and examples. All patents and publications mentioned herein (including all sequences disclosed in such patents and publications) are expressly incorporated by reference.

Justice

In general, unless otherwise defined, all technical terms, notations, and other scientific and technical terms used herein are intended to have the meaning commonly understood by a person skilled in the relevant fields of the present invention. For example, as used in phrases such as “A and/or B,” the term “and/or” means both A and B; A or B; A (single); and B (alone). Likewise, the term “and/or” used in phrases such as “A, B and/or C” is intended to include each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (alone).

As used herein, singular terms include their corresponding plural referents, unless the context clearly dictates otherwise.

As used herein, numerical ranges include the numbers that define the range.

The terms “about,” “approximately,” etc., when preceded by a list or range of numerical values, independently refer to each individual value in the list or range as if the term immediately preceded each individual value in the list or range. Indicates a value. The term means that a value exactly matches, is close to, or resembles the value to which it refers. For example, in some embodiments, about or approximately a particular value may represent a value of 99%, 95%, or 90% of the above value. By way of example, the term “about 100” includes 99 and 101, and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, 99.5, etc.). As another example, if the temperature is 70°C, “about” or “approximately” 70°C may be equivalent to 69°C, 66°C, or 63°C. It must be understood that this is merely a foreordination.

As used herein, nucleic acids are written 5' to 3' from left to right; Amino acid sequences are written from left to right, amino to carboxy. Practitioners should refer to Sambrook et al., 1989, and Ausubel FM et al., 1993 for technical definitions and terminology. It should be understood that the present invention is not limited to specific methodologies, protocols and reagents and may vary.

The terms “polypeptide,” “oligopeptide,” “peptide,” and “protein” are used interchangeably herein and refer to a chain of amino acids of any length, preferably relatively short (e.g., 10 to 100 amino acids). refers to The chain may be linear or branched, and/or it may contain modified amino acids and/or may be interrupted by non-amino acids. The term also refers to an amino acid chain that has been modified naturally or by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Includes. Also included within this definition are polypeptides comprising, for example, one or more analogs of amino acids (including, for example, unnatural amino acids, etc.) and other modifications known in the art. It is understood that polypeptides may occur as single chains or as associated chains. Preferably, mammalian polypeptides (polypeptides originally derived from mammalian organisms) are used, more preferably those secreted directly into the medium.

An “antibody” is an immunoglobulin molecule capable of specifically binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through one or more antigen recognition sites located in the variable region of the immunoglobulin molecule. As used herein, the terms include polyclonal antibody, monoclonal antibody, chimeric antibody, bispecific antibody, bi-specific antibody, bifunctional antibody, trispecific antibody, multispecific antibody, bispecific heterodimeric antibody. Bodies, bispecific heterodimeric IgG, labeled antibodies, humanized antibodies, human antibodies and fragments thereof (e.g. Fab, Fab', F(ab') ₂ , Fv), single chain (ScFv) and domain antibodies ( e.g. shark and camelid antibodies), fusion proteins comprising antibodies, and any other modified configuration of immunoglobulin molecules containing antigen recognition sites. Antibodies include antibodies of any class, such as IgG, IgA or IgM (or subclasses thereof), and the antibodies need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of the heavy chain, immunoglobulins can be assigned to different classes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, several of which are further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. You can. The heavy chain constant regions corresponding to the different classes of immunoglobulins are referred to as alpha, delta, epsilon, gamma, and mu, respectively. The subunit structure and three-dimensional arrangement of different classes of immunoglobulins are well known. The invention also includes “antibody analogs,” other non-antibody molecular protein-based scaffolds, such as fusion proteins and/or immunoconjugates that can provide specific antigen binding using CDRs. Antibodies of the invention may be derived from any species, including but not limited to mouse, human, camel, llama, fish, shark, goat, rabbit, chicken, horse, and cow.

The term "antibody" refers to an immunoglobulin molecule comprising four polypeptide chains, two heavy (H) and two light (L) chains interconnected by disulfide bonds, and multimers thereof (e.g. IgM). Included as. Each heavy chain includes a heavy chain variable region (abbreviated herein as HC VR or VH) and a heavy chain constant region. The “variable region” of an antibody refers to the variable region of the light chain of an antibody or the variable region of the heavy chain of an antibody, alone or in combination. The heavy chain constant region contains three domains: CH1, CH2 and CH3. CH1 and CH2 domains are connected by a hinge region. Each light chain comprises a light chain variable region (referred to herein as LC VR or VL) and a light chain constant region. The light chain constant region includes one domain (CL1). The VH and VL regions can be further divided into hypervariable regions, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). VH and VL each consist of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In various embodiments of the invention, the FR (or antigen binding portion thereof) of the CD3 antibody may be identical to the human germline sequence, or may be naturally or artificially modified. An amino acid consensus sequence can be defined based on side-by-side analysis of two or more CDRs. The CDRs of each chain are held together in close proximity by FRs and, together with the CDRs of the remaining chains, contribute to the formation of the antigen binding site of the antibody.

As used herein, the term “antigen-binding fragment,” “antibody fragment,” or “antigen-binding portion” refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments that fall within the term "antigen binding fragment" of an antibody include: (i) an antibody heavy chain variable domain (VH) and/or an antibody light chain variable domain (VL), or derived from a full-length antibody or antibody fragment; VH/VL pair, such as VH domain and/or VL domain; (ii) Fab fragment, which is a monovalent fragment consisting of VL, VH, CL and CH1 domains; (iii) Fab' fragments, which are essentially part of the Fab and hinge regions (e.g., Fundamental Immunology, Paul ed., 3.sup.rd ed. 1993); (iv), the F(ab') ₂ fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (v) consisting of VH and CH1 domains; (vi) an Fv fragment consisting of the VL and VH domains of a single arm of the antibody; (vii) single chain Fv fragment (scFv), which is a single protein chain in which the VL and VH regions pair to form a monovalent molecule (e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883]); (viii) a disulfide stabilized Fv fragment (dsFv), which is an Fv with an intermolecular disulfide bond engineered to stabilize the VH-VL pair; (ix) single variable domain antibody (sdAb or dAb) fragments consisting of the variable domain of the heavy chain and no light chain (e.g. Ward et al., (1989) Nature 341:544-546); (x) complementary determining region (CDR); and any derivative thereof.

As used herein, an “antigen-binding fragment” of an antibody may include one or more variable domains. A variable domain may be of any size or amino acid composition and will generally include one or more CDRs adjacent to or in frame with one or more framework sequences. As used herein, an “antigen-binding fragment of an antibody” refers to any of the variable and constant domains listed below in non-covalent association with each other and/or with one or more monomeric VH or VL regions (e.g., by disulfide bonds). It may include homodimers or heterodimers (or other multimers) in the array. For example, the variable region may be dimeric or contain VH-VH, VH-VL or VL-VL dimers. The array of variable and constant domains that can be found within the antigen-binding fragment of the antibody of the invention includes: VH-CH1; VH-CH2; VH-CH3; VH-CH1-CH2; VH-CH1-CH2-CH3; VH-CH2-CH3; VH-VL-CL, VH-VL-CH1, VH-VL-CH2; VH-CL; VL-CH1; VL-CH2; VL-CH3; VL-CH1-CH2; VL-CH1-CH2-CH3; VL-CH2-CH3; and VL-CL. The variable region and constant domain may be connected to each other directly or may be connected by a full or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids, which are located between adjacent variable and/or constant domains in a single polypeptide molecule. resulting in a flexible or semi-flexible connection.

As used herein, “binding domain” includes any region of a polypeptide (e.g., an antibody) that is responsible for selectively binding to a molecule of interest (e.g., an antigen, ligand, receptor, substrate, or inhibitor). Exemplary binding domains include antibody variable regions, receptor binding domains, ligand binding domains, and enzyme domains.

As used herein, the term “human acceptor framework” is a framework comprising the amino acid sequence of a light chain variable (VL) framework or a heavy chain variable (VH) framework derived from the human immunoglobulin framework or the human consensus framework, as defined below. . A human receptor framework “derived from” the human immunoglobulin framework or the human consensus framework may include the amino acid sequence thereof or may contain amino acid sequence modifications. In some embodiments, the number of amino acid modifications is no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, or no more than 2. In some embodiments, the VL human receptor framework is identical in sequence to a VL human immunoglobulin framework sequence or a human consensus framework sequence.

As used herein, an “affinity matured” antibody refers to an antibody that has one or more modifications in one or more variable regions (including CDRs and FRs) compared to a parent antibody that does not have the modifications, wherein the modifications are present on the antigen. It causes an improvement in the affinity of the antibody.

As used herein, the terms “Fc region”, “Fc domain”, “Fc chain” or similar terms are used to define the C-terminal region of an IgG heavy chain. The Fc region of IgG contains two constant domains, CH2 and CH3. The CH2 domain of the human IgG Fc region generally extends from amino acid 231 to amino acid 340 according to the numbering system of the EU index or from amino acid 244 to amino acid 360 according to the numbering system of Kabat. The CH3 domain of the human IgG Fc region generally extends from amino acid 341 to amino acid 447 according to the EU index numbering system or from amino acid 361 to amino acid 478 according to the Kabat numbering system. The CH2 domain of the human IgG Fc region (also referred to as the “Cγ 2” domain) is special in that it does not pair closely with another domain. Rather, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of intact native IgG. The Fc region may comprise native or variant Fc sequences. Although the boundaries of the Fc sequence of immunoglobulin heavy chains can vary, the human IgG heavy chain Fc sequence is generally defined as extending from amino acid residues around positions Cys226 or Pro230 to the carboxy terminus of the Fc sequence. Unless otherwise specified herein, amino acid residues of the Fc region or constant region are described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. According to the EU numbering system, also referred to as the EU Index, as described in Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

In certain embodiments, the Fc chain begins in the hinge region immediately upstream of the papain cleavage site and end and ends at the C-terminus of the antibody. Accordingly, a complete Fc chain includes at least a hinge domain, a CH2 domain, and a CH3 domain. In certain embodiments, the Fc chain comprises one or more of the following: a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, a CH4 domain, or a variant, portion, or fragment thereof. In certain embodiments, the Fc domain comprises a complete Fc chain (i.e., hinge domain, CH2 domain, and CH3 domain). In certain embodiments, the Fc chain comprises a hinge domain (or portion thereof) fused to a CH3 domain (or portion thereof). In certain embodiments, the Fc chain comprises a CH2 domain (or portion thereof) fused to a CH3 domain (or portion thereof). In certain embodiments, the Fc chain consists of a CH3 domain or portion thereof. In certain embodiments, the Fc chain consists of a hinge domain (or part thereof) and a CH3 domain (or part thereof). In certain embodiments, the Fc chain consists of a CH2 domain (or portion thereof) and a CH3 domain. In certain embodiments, the Fc chain consists of a hinge domain (or part thereof) and a CH2 domain (or part thereof). In certain embodiments, the Fc chain lacks at least a portion of the CH2 domain (e.g., all or part of the CH2 domain). Fc chain herein generally refers to a polypeptide comprising all or part of the Fc chain of an immunoglobulin heavy chain. This includes, but is not limited to, the entire CHI, hinge, CH2, and/or CH3 domains, and fragments of these peptides containing, for example, only the hinge, CH2, and CH3 domains. The Fc chain may be derived from any species of immunoglobulin and/or any subtype (including but not limited to human IgGl, IgG2, IgG3, IgG4, IgD, IgA, IgE or IgM antibodies). The Fc domain includes native Fc and Fc variant molecules. Like Fc variants and native Fc, the term Fc chain includes molecules in monomeric or multimeric form, whether digested from whole antibodies or produced by other means. In some embodiments, the Fc chain comprises the carboxy terminal portions of both heavy chains held together by disulfides. In certain embodiments, the Fc chain consists of a CH2 domain and a CH3 domain.

As used in the art, “Fc receptor” and “FcR” describe a receptor that binds to the Fc region of an antibody. The preferred FcR is a native sequence human FcR. Additionally, preferred FcRs are those that bind IgG antibodies (gamma receptors) and include receptors of the FcγRI, FcγRII and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA (“activating receptor”) and FcγRIIB (“inhibitory receptor”), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. FcR is described in Ravetch and Kinet, Ann. Rev. Immunol., 9:457-92, 1991]; [Capel et al., Immunomethods, 4:25-34, 1994]; and [de Haas et al., J. Lab. Clin. Med., 126:330-41, 1995. Additionally, “FcR” includes the neonatal receptor (FcRn), which is responsible for transferring maternal IgG to the fetus (Guyer et al., J. Immunol., 117:587, 1976); and Kim et al. , J. Immunol., 24:249, 1994]).

A “native sequence Fc region” or “wild-type Fc region” comprises an amino acid sequence identical to that of an Fc region found in nature. “Wild-type” human IgG Fc refers to the sequence of amino acids that occur naturally in the human population. Of course, the Fc sequence may differ slightly between individuals, but one or more changes may occur in the wild-type sequence and are nonetheless within the scope of the present invention. For example, the Fc region may contain additional alterations not relevant to the invention, such as mutations in glycosylation sites, inclusion of unnatural amino acids, or “knob-in-hole” mutations.

A “variant Fc region” or “variant Fc chain” may comprise an amino acid sequence that differs from that of the native sequence Fc region due to one or more amino acid modifications, but retains one or more effector functions of the native sequence Fc region. In some embodiments, a variant Fc chain has one or more amino acid substitutions, e.g., about 1 to about 10 amino acid substitutions, preferably about 1 to about 10 amino acid substitutions, compared to a native sequence Fc chain or compared to the Fc region of the parent polypeptide. It has five amino acid substitutions in the native sequence Fc chain or in the Fc chain of the parent polypeptide. The variant Fc chains herein preferably have at least about 80% sequence identity, most preferably, at least about 90% sequence identity, and more preferably, at least about 95% sequence identity to the native sequence Fc chain and/or the Fc chain of the parent polypeptide. %, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity.

As used herein, the term “effector function” refers to the biological activity attributable to the Fc chain of an antibody (native sequence Fc chain or amino acid sequence variant Fc chain) and may vary depending on the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; Downregulation of cell surface receptors (e.g. B cell receptor); and B cell activation. These effector functions generally require an Fc chain to be combined with a binding domain (e.g., an antibody variable region) and can be assessed by a variety of assays known in the art to assess such antibody effector functions. Exemplary measurements of effector function are performed through Fcγ3 and/or C1q binding.

The effector function of an antibody is determined by the sequence of its Fc chain; This chain is also the part recognized by the Fc receptor (FcR) found on certain types of cells.

In some embodiments, the Fc polypeptide comprises part or all of the wild-type hinge sequence (generally at its N-terminus). In some embodiments, the Fc polypeptide does not include a functional or wild-type hinge sequence.

As used herein, “hinge region”, “hinge sequence” and variations thereof are described, for example, in Janeway et al., ImmunoBiology: the immune system in health and disease, Elsevier Science Ltd., NY (4th ed., 1999). ]; [Bloom et al., Protein Science, 6:407-415, 1997]; and [Humphreys et al., J. Immunol. Methods, 209:193-202, 1997].

As used herein, “immunoglobulin-like hinge region”, “immunoglobulin-like hinge sequence” or variations thereof refer to the hinge region and hinge sequence of an immunoglobulin-like or antibody-like molecule (e.g., an immunoadhesin). do. In some embodiments, the immunoglobulin-like hinge region may originate or be derived from any IgG1, IgG2, IgG3 or IgG4 subtype, or from IgA, IgE, IgD or IgM (a chimeric form thereof, e.g., chimeric IgG1/ 2 hinge area).

In some embodiments, the hinge region may originate from a human IgG1 subtype extending from amino acid 216 to amino acid 230 according to the EU index numbering system or from amino acid 226 to amino acid 243 according to the Kabat numbering system. In some embodiments, the sequence may be EPKSCDKTHTCPPCP (SEQ ID NO: 63). Those skilled in the art may have different understandings of the exact amino acids that correspond to the various domains of the IgG molecule. Accordingly, the N-terminus or C-terminus of the domains outlined above may be extended or shortened by 1, 2, 3, 4, 5, 6, 7, 8, 9 or even 10 amino acids.

In some embodiments, the hinge region may be mutated by one or more amino acids. In some embodiments, the hinge region may be truncated or may contain only a portion of the entire hinge region. In some embodiments, the hinge region may contain only the last five amino acids of the hinge region, referred to herein as the “lower hinge” region. In some embodiments, the lower hinge region will consist of amino acid CPPCP (SEQ ID NO:64), amino acid 226 to amino acid 230 (according to the numbering system of the EU index), or amino acid 239 to amino acid 243 (according to Kabat's numbering system). You can.

As used herein, the term “modification” refers to amino acid substitutions, insertions, and/or deletions in a polypeptide sequence, alterations in moieties chemically linked to a protein, or modifications in the function of a protein, such as an antibody. For example, a modification may be an altered function of an antibody, or an altered carbohydrate structure attached to a protein. As used herein, “amino acid modification” refers to a mutation (substitution), insertion (addition), or deletion of one or more amino acid residues in an antibody. The term “amino acid mutation” refers to the substitution (e.g., replacement of an amino acid residue) of one or more existing amino acid residues by another different amino acid residue. The term “amino acid deletion” refers to the removal of one or more amino acid residues at a predetermined position in an amino acid sequence. For example, the mutation L234A represents a substitution of the amino acid residue lysine at position 234 of the antibody Fc-region by the amino acid residue alanine (substitution of lysine by alanine) (numbering scheme according to the EU index). “Naturally occurring amino acid residues” include alanine (3-letter code: Ala, 1-letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), and cysteine (Cys, C), glutamine (Gin, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (He, I), leucine (Leu, L), lysine (Lys, K) ), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y) and valine (Val, V).

The term “agent” is used herein to refer to a biological macromolecule, an extract produced from a biological material, a mixture of biological macromolecules, a chemical compound, a mixture of chemical compounds, and/or a mixture of a chemical compound and a biological macromolecule. The term “therapeutic agent” refers to an agent that has biological activity.

As used herein, “monoclonal antibody” refers to an antibody obtained from a population of antibodies that is substantially homogeneous, i.e., the individual antibodies making up the population are identical except for possible naturally occurring mutations that may be present in incidental amounts. Monoclonal antibodies are directed to a single antigenic site and are therefore highly specific. Additionally, in contrast to polyclonal antibody preparations, which typically include different antibodies directed to different determinants (epitopes), each monoclonal antibody is directed to a single determinant on the antigen. The modifier “monoclonal” refers to the characteristic of an antibody obtained from a substantially homogeneous population of antibodies and is not to be interpreted as requiring production of the antibody by any particular method. For example, the monoclonal antibodies used according to the invention can be produced by the hybridoma method first described in Kohler and Milstein, Nature 256:495, 1975, or by the recombinant DNA method described, for example, in US 4,816,567. can be produced by Monoclonal antibodies can also be isolated from phage libraries produced using techniques described, for example, in McCafferty et al., Nature 348:552-554, 1990.

Antibodies of the invention may be “humanized antibodies.” As used herein, a “humanized” antibody is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab', F(ab') ₂ or refers to a form of non-human (e.g., mouse, rat, rabbit, non-human primate or other mammalian) antibody that is a non-human (e.g., mouse, rat, rabbit, non-human primate or other mammalian) antibody. These non-human amino acid residues are commonly referred to as “import” residues, typically taken from an “import” variable domain. The importing moiety, sequence or antibody has the desired affinity and/or specificity, or other desired antibody biological activity, as discussed herein.

Preferably, the humanized antibody is comprised of residues from the complementarity determining region (CDR) of the recipient with residues from the CDR of a non-human species (donor antibody), such as mouse, rat or rabbit, with the desired specificity, affinity and potency. It is a replaced human immunoglobulin (recipient antibody). In some cases, Fv framework region (FR) residues of a human immunoglobulin are replaced with corresponding non-human residues. Additionally, humanized antibodies may contain residues that are not found in either the recipient antibody or the imported CDR or framework sequences, but are included to further improve and optimize antibody performance. Generally, a humanized antibody comprises substantially all of one or more, typically two, variable regions, wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions. is from the human immunoglobulin consensus sequence. Additionally, humanized antibodies will optimally comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Antibodies with a modified Fc chain as described in WO 99/58572 are preferred. Other forms of humanized antibodies include one or more CDRs (CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, or CDR H3) that have been altered relative to the original antibody (one or more CDRs that are "derived" from one or more CDRs from the original antibody). (also referred to as ). As used herein, “humanized” is intended to include antibodies that have been deimmunized.

As used herein, “human antibody” refers to an antibody having an amino acid sequence that corresponds to that of an antibody produced by humans and/or produced using any of the human antibody production techniques known to those skilled in the art or disclosed herein. Accordingly, as used herein, the term “human antibody” is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the invention may contain, for example, amino acid residues not encoded by human germline immunoglobulin sequences in the CDRs, particularly CDR3 (e.g., introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). mutations) may be included. This definition of human antibody includes antibodies comprising one or more human heavy chain polypeptides or one or more human light chain polypeptides. One example thereof is an antibody comprising murine light chain and human heavy chain polypeptides. Human antibodies can be produced using a variety of techniques known in the art. In one embodiment, the human antibody is selected from a phage library, wherein the phage library expresses a human antibody (Vaughan et al., Nature Biotechnology, 14:309-314, 1996; Sheets et al., Proc. Natl. Acad. Sci. (USA) 95:6157-6162, 1998]; [Hoogenboom and Winter, J. Mol. Biol., 227:381, 1991]; [Marks et al., J. Mol. Biol. ., 222:581, 1991]). Human antibodies can also be produced by immunization of animals into which human immunoglobulin loci have been introduced by transgenics in place of the endogenous loci, for example, mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described in US 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016. Alternatively, human antibodies can be produced by immortalizing human B lymphocytes that produce antibodies directed to the target antigen (such B lymphocytes can be recovered from an individual or from single cell cloning of cDNA, or immunized in vitro). there is). For example, Cole et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77, 1985]; [Boerner et al., J. Immunol., 147(1):86-95, 1991]; and US 5,750,373.

Human antibodies of the invention may exist in two or more forms associated with hinge heterologous components. For example, immunoglobulin molecules comprise stable four chain constructs of about 150 to 160 kDa whose dimers are held together by interchain heavy chain disulfide bonds. Alternatively, dimers are molecules of about 75 to 80 kDa (half-antibodies) formed to consist of covalently coupled light and heavy chains, not linked through interchain disulfide bonds.

As used herein, the term “recombinant human antibody” refers to any human antibody produced, expressed, produced and isolated by recombinant methods, such as antibodies expressed using recombinant expression vectors transfected into host cells (described further below). , antibodies isolated from recombinant conjugated human antibody libraries (described further below), antibodies isolated from animals (e.g. mice) transgenic for human immunoglobulin genes (e.g. Taylor et al. 1992) Nucl. Acids Res. 20:6287-6295], or any other method that involves splicing a human immunoglobulin gene sequence to another DNA sequence. It is intended to include. These recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are mutagenized in vitro (or, if animal transgenics for human Ig sequences are used, somatically mutagenized in vivo), and thus amino acids in the VH and VL regions of the recombinant antibody. Sequences are sequences that are derived from and are related to human germline VH and VL sequences but do not naturally exist within the human antibody germline repertoire in vivo.

Antibodies of the invention may have part of the heavy and/or light chain derived from a particular species or identical to or corresponding to the corresponding sequence of an antibody belonging to a particular antibody class or subclass, and the remainder of the chain being derived from another species. Antibodies belonging to another antibody class or subclass, and identical or equivalent sequences of fragments of such antibodies, may be "chimeric" antibodies, which exhibit the desired biological activity (US 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81 :6851 -6855, 1984]). Chimeric antibodies of interest herein include primatized antibodies comprising variable region antigen binding sequences derived from non-human primates (e.g., Old World monkeys, apes, etc.) and human constant region sequences.

“Monovalent antibodies” contain one antigen binding site per molecule (e.g., IgG or Fab). In some cases, a monovalent antibody may have more than one antigen binding site, but the binding sites are from different antigens.

“Monospecific antibody” refers to an antibody or antibody preparation containing two identical antigen binding sites per molecule (e.g., IgG), where the two binding sites bind the same epitope on the antigen. Therefore, they compete with each other for binding to one antigen molecule. The term includes “monoclonal antibody” or “monoclonal antibody composition”. Most antibodies found in nature are monospecific. In some cases, monospecific antibodies may also be monovalent antibodies (e.g. Fab).

The terms “mutational burden”, “mutagenic burden”, “mutational burden” and “mutational burden” are used interchangeably herein. Tumor mutation load is an indication of the number of mutations in the tumor genome, defined as the total number of mutations per coding region of the tumor genome. There is large variability in mutational burden within tumor types, ranging from just a few to thousands of mutations (Alexandrov LB et al., Nature 2013;500(7463):415-421; Lawrence MS et al., Nature 2013;499:214-218]; [Vogelstein B et al., Science, 2013;339:1546-1558]).

The “original amino acid” residue is replaced with an “import amino acid” residue that may have a smaller or larger side chain volume than the original residue. The imported amino acid residue may be a naturally occurring or non-naturally occurring amino acid residue, but is preferably the former. “Naturally occurring” amino acid residues are those encoded by the genetic code. A “non-naturally occurring” amino acid residue is a residue that is not encoded by the genetic code, but which can be covalently linked to adjacent amino acid residues in the polypeptide chain. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine, and other amino acid residue analogs, such as those described in Ellman et al., Meth. Enzyme. 202:301-336 (1991)]. In some embodiments, the methods of the invention involve replacement of one or more original amino acid residues, although more than one original residue may be replaced. In general, no more than the total residues of the junction of the first or second polypeptide may comprise the original amino acid residue being replaced.

As used herein with reference to an antibody, the term "compete" means that a first antibody, or antigen-binding fragment (or portion) thereof, binds to an epitope in a manner sufficiently similar to the binding of a second antibody, or antigen-binding portion thereof, that the first antibody means that the result of binding of a cognate epitope to a cognate epitope is noticeably reduced in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. An alternative is that the binding of the second antibody to its epitope is also noticeably reduced in the presence of the first antibody, although this may be the case. That is, the first antibody can inhibit the binding of the second antibody to its respective epitope in the absence of the second antibody that inhibits the binding of the first antibody to its respective epitope. However, antibodies are said to "cross-compete" with each other for binding of their respective epitopes if each antibody significantly inhibits the binding of another antibody to its cognate epitope or ligand to the same, greater or lesser extent. It is said. Both competitive and cross-competitive antibodies are encompassed by the invention. Regardless of the mechanism by which competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or portion thereof), the skilled artisan will be able to determine, based on the teachings provided herein, competition and/or cross-competition. It is understood that antibodies are included and may be useful in the methods disclosed herein.

As used herein, “antibody dependent cell mediated cytotoxicity” or “ADCC” refers to non-specific cytotoxic cells (such as natural killer (NK) cells, neutrophils, and macrophages) expressing Fc receptors (FcRs) targeting target cells. Refers to a cell-mediated reaction that recognizes an antibody bound to the target cell and subsequently causes lysis of the target cell. The ADCC activity of the molecule can be assessed using an in vitro ADCC assay as described in US 5,500,362 or 5,821,337. Useful effector cells for this assay include peripheral blood mononuclear cells (PBMC) and NK cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be assessed in vivo in animal models, such as those disclosed in Clynes et al., PNAS (USA), 95:652-656, 1998.

As used herein, “complement dependent cytotoxicity” or “CDC” refers to lysis of a target in the presence of complement. The complement activation pathway is initiated by binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) complexed with its cognate antigen. To assess complement activation, see, e.g., Gazzano-Santoro et al., J. Immunol. The CDC analysis described in Methods, 202: 163, 1996 can be performed.

As used herein, the terms “immune specifically bind,” “immune specifically recognize,” “specifically bind,” “specifically recognize,” and similar terms refer to a molecule, e.g., a binding domain. This means that it binds specifically to an antigen (e.g. an epitope or immune complex) but does not specifically bind to another molecule. Molecules that specifically bind to an antigen may bind other peptides or polypeptides with low affinity as determined by assays known in the art, such as immunoassays, BIACORE™ or other assays. Preferably, the molecule that specifically binds to the antigen does not cross-react with other proteins.

Suitable “moderate stringency conditions” include prewashing in a solution of 5 Hybridize overnight in 5×SSC at 50°C to 65°C; followed by two washes at 65°C for 20 minutes each with 2X, 0.5X and 0.2X SSC containing 0.1% SDS.

As used herein, “highly stringent conditions” or “high stringency conditions” refer to (1) low ionic strength and high temperature during washing, e.g., 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate; is used at 50°C, or (2) during hybridization a denaturing agent such as formamide, e.g. 50% containing 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer. (v/v) formamide (pH 6.5) was used with 750 mM sodium chloride, 75 mM sodium citrate at 42°C, or (3) with washing in 0.2x SSC (sodium chloride/sodium citrate) at 42°C. 50% formamide, 5x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% pyrosodium phosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50 μg/mL ), 0.1% SDS and 10% dextran sulfate at 42°C, and 50% formamide at 55°C, followed by a high-stringency wash consisting of EDTA with 0.1x SSC at 55°C. Those skilled in the art will know how to adjust temperature, ionic strength, etc. as needed to accommodate factors such as probe length, etc.

Binding protein, binding domain, CDR or antibody (broadly defined herein) includes the following definitions: Kabat definition, Chothia definition, accumulation of both Kabat and Chothia definition, AbM definition, contact definition, It may be identified according to North definition and/or shape definition, or any CDR determination method. See, for example, Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th ed. (hypervariable regions)]; See [Chothia et al., Nature 342:877-883, 1989 (structural loop structures)]. The identity of the amino acid residues of a particular antibody that make up the CDR can be determined using methods well known in the art. The AbM definition in CDR is a compromise between Kabat and Chotia and uses Oxford Molecular's AbM antibody modeling software (Accelrys®). The definition of “contact” in CDR is found in MacCallum et al., J. Mol. Biol., 262:732-745, 1996]. The “conformation” definition of a CDR is based on the residues contributing enthalpy to antigen binding (see, e.g., Makabe et al., J. Biol. Chem., 283:1156-1166, 2008). North identified basic CDR conformations using various preferred sets of CDR definitions (North et al., J. Mol. Biol. 406: 228-256, 2011). In another approach, referred to herein as “conformational definition” of a CDR, the position of the CDR can be identified as the residue contributing enthalpy to antigen binding (Makabe et al., J Biol. Chem. 283:1156- 1166, 2008]). Alternative CDR boundary definitions do not strictly follow either of the above approaches, but can be shortened or lengthened to take into account predictions or experimental results that specific residues or groups of residues, or even entire CDRs, do not significantly affect antigen binding, but Kabat It will overlap at least part of the CDR. As used herein, CDR may refer to a CDR defined by any approach known in the art (including combinations of approaches). The methods used herein may use CDRs defined according to any of these approaches. For any given embodiment containing more than one CDR, the CDR (or other residues of the antibody) is Kabat, Chotia, Expanded Definition (Kabat and Chotia), North, Expanded Definition, AbM, Contact. , and/or may be defined according to any of the form definitions.

The residues of the variable domain are numbered according to Kabat, the numbering system used for the heavy or light chain variable regions of a compilation of antibodies. Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. Using this numbering system, a truly linear amino acid sequence may contain a few or additional amino acids that correspond to shortenings or insertions into the FR or CDR of the variable region. For example, the heavy chain variable region may have a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and a residue insertion after residue 82 of heavy chain FR (e.g. residues 82a, 82b and 82c according to Kabat). may include. The Kabat numbering of residues can be determined for a given antibody by alignment in homologous regions of the antibody's sequence to a “standard” Kabat numbered sequence. Various algorithms for the imposition of Kabat numbering are available. The algorithm used in Abysis' version 2.3.3 release (www.abysis.org) was used to impose Kabat numbering on variable regions VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3. Additionally, specific amino acid residue positions in an antibody may be numbered according to Kabat.

As used herein, the term “phage display library” refers to a population of bacteriophages, each of which contains foreign cDNA recombinantly fused in frame to a surface protein. Phages display foreign proteins encoded by cDNA on their surface. After replication in a bacterial host (typically Eschericia coli ), phages containing the foreign cDNA of interest are selected by expression of the foreign protein on the phage surface.

The term “epitope” refers to a portion of a molecule that can be recognized by an antibody and/or create contacts and/or bind to one or more of the antigen binding regions of the antibody, known as paratopes. . A single antigen may have more than one epitope. Therefore, different antibodies may bind different regions on the antigen and have different biological effects. Epitopes often consist of molecules labeled with a chemically active surface, such as amino acids or sugar side chains, and have specific three-dimensional structural features and specific charge characteristics. As used herein, epitopes may be conformational epitopes or linear epitopes. Conformational epitopes are produced by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are those produced by adjacent amino acid residues in a polypeptide chain. In certain embodiments, an epitope may comprise a moiety of a saccharide, phosphoryl group, or sulfonyl group on the antigen.

As used herein, the term “antigenic epitope” is defined as the portion of a polypeptide to which an antibody specifically binds, as determined by any method well known in the art, for example, by conventional immunoassays. A “non-linear epitope” or “conformational epitope” comprises a non-contiguous polypeptide (or amino acid) within an antigenic protein to which an antibody specific for the epitope binds. After the desired epitope on an antigen has been determined, it is possible to produce antibodies against that epitope, for example using the techniques described herein.

The terms "specific binding" or "specific binding" when used in connection with the interaction of an antibody with a protein or peptide refers to an interaction that is dependent on the presence of a specific structure (i.e. an antigenic determinant or epitope) on the protein. ; In other words, antibodies generally recognize and bind to specific protein structures rather than proteins. For example, if an antibody is specific for epitope "A", the presence of a protein containing epitope A (or free unlabeled A) in a reaction containing labeled "A" and the antibody will determine whether the label bound to the antibody The amount of A will be reduced.

In certain embodiments, “specifically binds” means that the antibody binds to a protein, for example, with a K _D of less than about 0.1 nM, more typically less than about 1 μM. In certain embodiments, “specifically binds” means that the antibody binds to the target with a K _D of at least about 0.1 μM or less, at other times at least about 0.01 μM or less, and at other times at least about 1 nM or less. Due to sequence identity between homologous proteins in different species, specific binding may involve antibodies that recognize proteins from more than one species (e.g., human and mouse proteins). Likewise, due to homologous relationships within certain regions of the polypeptide sequences of different proteins, specific binding may involve antibodies that recognize more than one protein. It is understood that in certain embodiments, an antibody or binding moiety that specifically binds a first target may not or may specifically bind a second target. As such, “specific binding” does not necessarily require (but may include) exclusive binding, i.e., binding to a single target. Accordingly, in some embodiments, an antibody may specifically bind more than one target. In certain embodiments, multiple targets can be bound by the same antigen binding site on an antibody. For example, an antibody may, in certain instances, comprise two identical antigen binding sites, each of which specifically binds to the same epitope on two or more proteins. In certain alternative embodiments, an antibody may be multispecific and may comprise two or more antigen binding sites with different specificities. As a non-limiting example, a bispecific antibody may comprise one antigen binding site that recognizes an epitope on one protein (e.g., human CD3) and a second, different antigen binding site that recognizes a different epitope on a second protein. It may additionally include an antigen binding site. Generally, but not necessarily, reference to binding means specific binding.

Antibodies that specifically bind to an antigen do not bind other peptides or polypeptides due to low affinity as determined by assays known in the art, such as immunoassays, BIACORE® or other assays. Preferably, an antibody that specifically binds to an antigen does not cross-react with other proteins.

When used in connection with the interaction of an antibody with a protein or peptide, the terms "non-specific binding" or "background binding" refers to an interaction that is not dependent on the presence of a specific structure (i.e., an antibody binds to a specific structure, such as an epitope, rather than an epitope). usually binds to proteins).

As used herein, the term “k _on ” or “k _a ” refers to the rate constant of binding of an antibody to an antigen. Specifically, rate constants (k _on /k _a and k _off /k _d ) and equilibrium dissociation constants are measured using whole antibodies (i.e. bivalent) and monomeric CD3 protein.

As used herein, the term “k _off ” or “k _d ” refers to the rate constant of dissociation of an antibody from an antibody/antigen complex.

As used herein, the term “K _D ” refers to the equilibrium dissociation constant of antibody-antigen interaction.

As used herein, the term “binding affinity” generally refers to the strength of the sum of the total non-covalent interactions between a single binding site on a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, “binding affinity” as used herein refers to intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for partner Y can generally be expressed in terms of the dissociation constant (K _D ). The affinity of molecule X for partner Y can generally be expressed in terms of the dissociation constant (Kd). For example, Kd is about 200 nM, 150 nM, 100 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 8 nM, 6 nM, 4 nM, 2 nM, 1 nM or more. You can be strong. Affinity can be measured by routine methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen rapidly and tend to remain bound longer. A variety of methods for measuring binding affinity are known in the art, any of which may be used for the purposes of the present invention. In particular, the term “binding affinity” is intended to refer to the rate of dissociation of a particular antigen-antibody interaction. K _D is the ratio of the “dissociation rate (k _off )” to the “association rate (k _on )”. Therefore, K _D is equal to k _off /k _on and is expressed as molar concentration (M). Therefore, the smaller K _D, the stronger the binding affinity. Therefore, a K _D of 1 μM shows a weak binding affinity compared to a K _D of 1 nM. The K _D value of an antibody can be determined using methods established in the art. One way to determine the K _D of an antibody is by using surface plasmon resonance (SPR), typically using a biosensor system, such as the BIACORE™ system. BIACORE™ kinetic analysis involves analyzing the association and dissociation of antigens from a chip with molecules (e.g., molecules comprising an epitope binding domain) immobilized on the surface. Another way to determine the K _D of an antibody is by using Bio-Layer interferometry, typically using OCTET® technology (Octet QKe system (ForteBio)).

“Biologically active,” “biological activity,” and “biological properties” with respect to an antibody of the invention, such as an antibody, fragment or derivative thereof, means having the ability to bind to a biological molecule, except where specified.

As used herein, the terms “nucleic acid” and “nucleotide sequence” refer to DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), combinations of DNA and RNA molecules or hybrid DNA/RNA molecules, and DNA molecules. or analogs of RNA molecules. Such analogs can be produced using, for example, nucleotide analogs, including but not limited to inosine or tritylated bases. Additionally, such analogs may include DNA or RNA molecules that contain a modified backbone that provides the molecule with advantageous properties, such as nuclease resistance or increased ability to cross cell membranes. The nucleic acid or nucleotide sequence may be single- or double-stranded, may contain both single- and double-stranded portions, and may contain triple-stranded portions, but is preferably double-stranded DNA.

The invention also includes antibodies of the invention, including polypeptides, and polynucleotides encoding the binding regions of the antibodies. Polynucleotides encoding molecules of the invention can be obtained, and the nucleotide sequence of the polynucleotide is determined by any method known in the art.

A polynucleotide encoding an antibody of the invention may comprise: a coding sequence for only the variant, a coding sequence for the variant and additional coding sequences such as a functional polypeptide, or signal or secretion sequence or pre-protein sequence; Coding sequences and non-coding sequences for the antibody, such as introns, or non-coding sequences 5' and/or 3' of the coding sequence for the antibody. The term “polynucleotide encoding an antibody” includes polynucleotides comprising additional coding sequences for variants as well as polynucleotides comprising additional coding and/or non-coding sequences. It is known in the art that polynucleotide sequences optimized for a specific host cell/expression system can be readily obtained from the amino acid sequence of the protein of interest (see GENEART® AG, Regensburg, Germany). ).

Antibodies of the invention may have additional conservative or non-essential amino acid substitutions that do not substantially affect polypeptide function. Whether a particular substitution is tolerable, i.e. does not negatively affect the desired biological property, such as binding activity, can be determined by looking at Bowie, JU et al. Science 247: 1306-1310, 1990] or [Padlan et al. FASEB J. 9: 133-139, 1995.

As used herein, a “conservative amino acid substitution” is one in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are defined herein. These families include basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. asparagine, glutamine, serine, threonine, tyrosine, cysteine), Nonpolar side chains (e.g. glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine), and aromatic side chains (e.g. tyrosine, phenylalanine) , tryptophan, and histidine).

As used herein, a “non-essential” amino acid residue is a residue that can be altered from a binder, e.g., an antibody, of the wild-type sequence without eliminating or substantially altering its biological activity, whereas an “essential” amino acid residue is one in which such change is not made. cause In antibodies, essential amino acid residues may be specificity determining residues (SDRs).

As used herein, the term “isolated” refers to a material that has been removed from its original environment (e.g., the natural environment if naturally occurring). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but a polynucleotide or polypeptide separated from some or all of the materials that coexist in nature is isolated. Such polynucleotide may be part of a vector and/or such polynucleotide or polypeptide may be part of a composition, such as a mixture, solution or suspension, comprising isolated cells or cultured cells comprising the polynucleotide or polypeptide, A vector or composition may be isolated in that it is not part of its natural environment. For example, a polypeptide that is chemically synthesized or synthesized in a cell system different from the cell from which the polypeptide is naturally derived will be “isolated” from its naturally associated components. Additionally, the protein can be substantially free of naturally associated components by isolation using protein purification techniques well known in the art.

An “isolated” or “purified” antibody is one that, when chemically synthesized, is substantially free of cellular material or other contaminating proteins from a cell or tissue source, or the medium from which the proteins are derived, or chemical precursors or other chemicals. It does not contain substantially any. The term “substantially free of cellular material” includes preparations of antibodies in which the polypeptide/protein is separated from the cellular components of the cell from which it was isolated or recombinantly produced. Accordingly, an antibody substantially free of cellular material is a preparation of an antibody having less than about 50%, 40%, 30%, 20%, 10%, 5%, 2.5%, or 1% (by dry matter weight) contaminating protein. Includes. When the antibody is produced recombinantly, it is also preferred to be substantially free of culture medium, i.e. the culture medium represents about 50%, 40%, 30%, 20%, 10%, 5%, 2.5% of the volume of the protein preparation. It represents % or less than 1%. When an antibody is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals and reagents, i.e., the antibody is separated from the chemical precursors or other chemicals involved in the synthesis of the protein. Accordingly, preparations of such antibodies have less than about 50%, 40%, 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or compounds other than the antibody of interest. In a preferred embodiment of the invention, the antibody is isolated or purified. An antibody that has been isolated or removed from a component of one or more organisms, or from the tissues or cells in which the antibody exists or is naturally produced, is an “isolated antibody” for the purposes of the present invention.

As used herein, the term “recovery” refers to a process by which a chemical species, such as a polypeptide, is rendered substantially free of naturally associated components, for example, by isolation using protein purification techniques well known in the art. .

As used herein, the term “replicon” refers to any genetic element, such as a plasmid, chromosome, or virus, that acts as an autonomous unit of polynucleotide replication within a cell.

As used herein, the term “operably linked” refers to a situation in which the described components are in a relationship such that they function in their intended manner. For example, a control sequence that is “operably linked” to a coding sequence is omitted in such a way that expression of the coding sequence is achieved under conditions that are suitable or compatible with the control sequence. Generally, “operably linked” means that the DNA sequences being linked are contiguous and, in the case of secretory leaders, are contiguous and in translation. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are conventionally used.

As used herein, “vector” refers to a construct capable of delivering, and preferably expressing, one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and Includes certain eukaryotic cells, such as producer cells.

As used herein, the term “expression control sequence” or “control sequence” refers to a polynucleotide sequence necessary to effect expression of the coding sequence to which it is ligated. The nature of these control sequences varies depending on the host organism. For example, in prokaryotes, these control sequences typically include promoters, ribosome binding sites and terminators, and in some cases, enhancers. Accordingly, the term “control sequence” is intended to include at least all elements whose presence is required for expression, and may also include additional elements whose presence is advantageous, such as a leader sequence.

“Host cell” includes an individual cell or cell culture that can be or has been a recipient for a vector for incorporation of a polynucleotide insert. A host cell includes the progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or genomic DNA complementarity) to the original parent cell due to natural, accidental, or intentional mutations. Host cells include cells transfected in vivo with a polynucleotide of the invention.

As used herein, “mammalian cell” includes cells derived from mammals, including humans, rats, mice, guinea pigs, chimpanzees, or macaques. Cells can be cultured in vivo or in vitro.

As used herein, the term “purified product” refers to a preparation of the product that has been isolated from cellular components with which the product is normally associated and/or from other types of cells that may be present in the sample.

As used herein, “substantially pure” means at least 50% pure (i.e. free of contaminants), more preferably at least 90% pure, more preferably at least 95% pure, even more preferably at least 98% pure, Most preferably it refers to a material that is at least 99% pure.

As used herein, the terms “treat,” “treating,” or “treatment” refer to an approach that results in a beneficial or desired clinical outcome. For the purposes of the present invention, treatment refers to the administration of a CD3 antibody molecule (e.g. monoclonal antibody, bispecific) to an individual, e.g. a patient, or isolated tissue or cells from the individual e.g. returned to the individual. It may be administered by application to. CD3 antibody molecules can be administered alone or in combination with one or more agents. Treatment may be to cure, cure, alleviate, relieve, change, fix, improve, alleviate or affect the disorder, the symptoms of the disorder or the predisposition to the disorder, such as cancer.

As used herein, the term “individual” is intended to include any animal (e.g., mammal), including but not limited to humans, non-human primates, rodents, etc., that may be recipients of a particular treatment. For example, the subject may be a patient suffering from cancer (e.g., a human patient or a veterinary patient). Typically, the terms “individual,” “subject,” and “patient” are used interchangeably with respect to human subjects.

The term "non-human animal" as used herein, unless otherwise specified, includes all non-human vertebrates, including non-human mammals and non-mammals, such as non-human primates, sheep, dogs, horses, and chickens. , amphibians, reptiles, mice, rats, rabbits or goats, etc.

As used herein, the term “pharmaceutically acceptable” means approved (approvable) by a federal or state regulatory agency for use in animals, including humans, or approved by the US Pharmacopoeia or other generally accepted Refers to a product or compound listed in the Pharmacopoeia.

As used herein, the term “pharmaceutically acceptable excipient, carrier or adjuvant” or “acceptable pharmaceutical carrier” refers to an excipient that can be administered to an individual with one or more antibodies of the present disclosure and does not destroy the activity of the antibody, Refers to a carrier or auxiliary agent. Excipients, carriers or adjuvants should be non-toxic when administered with the antibody in a dose sufficient to deliver a therapeutic effect.

As used herein, the term “improvement” means relief or improvement of one or more symptoms compared to not administering an antibody molecule of the invention. Additionally, “improvement” includes shortening or shortening the duration of symptoms.

As used herein, the terms “prevent,” “preventing,” and “prophylaxis” refer to the prevention of recurrence or onset of one or more symptoms of a disorder in an individual as a result of administration of a prophylactic or therapeutic agent.

As used herein, “effective amount,” “therapeutically effective amount,” “therapeutically sufficient amount,” or “effective dose” refers to an amount that, when administered to a subject in a single or multiple doses, is greater than would be expected in the absence of such treatment. , effective in preventing, curing, ameliorating, treating or managing a disease, disorder or adverse event, or reducing the rate of development of a disease or disorder, or prolonging the cure, alleviation, alleviation or amelioration of the condition of an individual with a disorder described herein. or refers to an amount of therapeutic agent (e.g., a CD3 antibody alone or as part of a multispecific antibody (e.g., bispecific)) that is sufficient. Additionally, the term includes within its range amounts effective to improve general physiological function. An effective amount may be considered in the administration of one or more therapeutic agents, and a single agent may be considered to be provided in an effective amount if it achieves or is capable of achieving the desired result in combination with one or more other agents.

As used herein, an effective amount can target effector T cells to induce T cell mediated cytotoxicity of cells associated with a disease, such as cancer, an autoimmune disease, or an infectious disease. The therapeutic agents of the present invention can induce a T cell mediated immune response by modulating CD3 antigens on the surface of T cells, particularly CD3, more particularly CD3 epsilon.

With respect to cancer, a therapeutically effective amount refers to the amount of a therapeutic agent that inhibits the growth of a tumor or cancer by slowing, blocking, arresting or stopping the growth and/or metastasis of the tumor or cancer.

Efficacy is a measure of the activity of a therapeutic agent expressed in the amount needed to produce an effect of a given intensity. High potency agents cause a greater response at low concentrations compared to low potency agents which cause less response at low concentrations. Efficacy is a function of affinity and potency. Potency refers to the ability of a therapeutic agent to produce a biological response upon binding to a target ligand and the quantitative magnitude of that response. As used herein, the term “semimaximal effective concentration (EC ₅₀ )” refers to the concentration of therapeutic agent that causes a response intermediate between the basal and maximal values after a specified exposure time. Therapeutic agents can cause inhibition or stimulation. The EC ₅₀ value is commonly used and used herein as a measure of efficacy.

As used herein, “combination therapy” or “administration in combination” refers to the use of more than one prophylactic and/or therapeutic agent. Use of the terms “combination therapy” or “in combination” does not limit the order in which prophylactic and/or therapeutic agents are administered to an individual suffering from a disorder. In other words, combination therapy can be performed by treating the therapeutic agents individually, sequentially or simultaneously. In the case of “sequential administration,” the first administered agent may exert some physiological effect on the subject when the second agent is administered or activated in the subject.

As used herein in relation to the administration of prophylactic and/or therapeutic agents, the term “simultaneous administration” refers to the administration of agents such that separate agents are present simultaneously in an individual. Simultaneous administration can be accomplished by molecules formulated in a single composition, or in separate compositions administered simultaneously or at similar times. Sequential administration can be performed in any order as needed.

The term “cluster of differentiation 3” or “CD3” refers to a multimeric protein complex (known as the T3 complex), consisting of four distinct polypeptide chains: epsilon (ε), gamma (γ), delta (δ), and zeta (ζ). ), which combines and functions as three pairs of dimers (εγ, εδ, ζζ). The CD3 complex acts as a T cell co-receptor that non-covalently associates with the T cell receptor (TCR) (Smith-Garvin et al. 2009) and generates an activation signal in T lymphocytes. The CD3 protein complex is an essential defining feature of the T cell lineage, and therefore CD3 antibodies can be effectively used as T cell markers (Chetty and Gatter, Journal of Pathology, Vol 172, 4, 303-301 (1994) ). It is known that CD3 antibodies can induce the production of cytotoxic T cells and selectively kill tumor targets through activation of endogenous lymphokine production (Yun et al., Cancer Research, 49:4770-4774 , 1989]).

T cells play a central role in cell-mediated immunity in humans and animals. Recognition and binding of specific antigens is mediated by TCRs expressed on the surface of T cells. The TCR of T cells interacts with immunogenic peptides (epitopes) that bind to major histocompatibility complex (MHC) molecules and are presented on the surface of target cells. Specific binding to the TCR triggers a signaling cascade within the T cell, resulting in proliferation and differentiation into mature effector T cells. More specifically, T cells express TCR complexes that can induce antigen-specific immune responses (Smith-Garvin et al, 2009). Antigens are peptides expressed by tumor cells and virally infected cells that can stimulate an immune response. Antigens expressed within cells bind to major histocompatibility class I (MHC class I) molecules and move to the surface exposed to T cells. If the binding affinity of the TCR for MHC class I in complex with antigen is sufficient, the formation of an immune synapse begins. Signaling across the immune synapse is mediated through CD3 co-receptors that form epsilon/delta, delta/gamma and zeta/zeta dimers. These dimers associate with the TCR and generate an activation signal in T lymphocytes. This signaling cascade leads to T cell-mediated killing of cells expressing the antigen. Cytotoxicity is mediated by the release and translocation of granzyme B and perforin from T cells to target cells.

As used herein, “activating T cell antigen” refers to an antigenic determinant expressed on the surface of a T lymphocyte, especially a cytotoxic T lymphocyte, that can induce T cell activation upon interaction with an antigen binding molecule. Specifically, the interaction of an antigen-binding molecule with an activated T cell antigen can induce T cell activation by triggering a signaling cascade of the T cell receptor complex. In certain embodiments, the activating T cell antigen is CD3, particularly the epsilon subunit of CD3 (see Uniprot No. P07766 (version 130), NCBI RefSeq No. NP_000724.1, SEQ ID NO: 66 for the human sequence). Typically, the naturally occurring allelic variant has an amino acid sequence that is at least 95%, 97% or 99% identical to the protein described in Accession No. BAB71849.1.

As used herein, “T cell activation” refers to one or more cellular responses of T lymphocytes, especially cytotoxic T lymphocytes, selected from proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity and expression of activation markers. . The T cell activating bispecific antigen binding molecule of the present invention can induce T cell activation. Suitable assays for measuring T cell activation are known in the art, as described herein.

As used herein, “antibody that binds CD3,” “antibody that recognizes CD3,” “anti-CD3 antibody,” or “CD3 antibody” refers to a single CD3 subunit (e.g., epsilon, delta, gamma, or zeta). Antibodies and antigen-binding fragments thereof that specifically recognize dimeric complexes of two CD3 subunits (e.g., gamma/epsilon, delta/epsilon, and zeta/zeta CD3 dimers) and antigens thereof Contains a binding fragment. Human CD3 epsilon is shown in GenBank accession number NM_000733. Antibodies of the invention can bind soluble CD3 and/or cell surface expressed CD3. Soluble CD3 includes the native CD3 protein and recombinant CD3 protein variants that lack the transmembrane domain or do not associate with the cell membrane, such as monomeric and dimeric CD3 constructs.

CD3-directed antibodies can generate activation signals in T lymphocytes. Other T cell activating ligands may also be used, including but not limited to CD28, CD134, CD137, and CD27.

The present invention includes one-arm antibodies that bind CD3. As used herein, “one-arm antibody” refers to an antigen binding molecule comprising a single antibody heavy chain and a single antibody light chain. One-arm antibodies of the invention may comprise any of the VL/VH or CDR amino acid sequences shown in Tables 1, 2 and 3 herein.

The CD3 antibodies disclosed herein may contain one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequence from which the antibody is derived. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The invention includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, wherein at least one amino acid in one or more framework and/or CDR regions is a corresponding residue of the germline sequence from which the antibody is derived, and Mutated to a corresponding residue in another human germline sequence, or by a conservative amino acid substitution of a corresponding germline residue (such sequence changes are collectively referred to herein as “germline mutations”). One skilled in the art can readily produce many antibodies and antigen-binding fragments containing one or more distinct germline mutations or combinations thereof starting from the heavy and light chain variable region sequences disclosed herein. In certain embodiments, all framework and/or CDR residues within the VH and/or VL domains are back mutated to residues found in the original germline sequence from which the antibody was derived. In some embodiments, only certain residues are mutated, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2, or CDR3. Mutations occur back to the original germline sequence. In some additional embodiments, one or more of the framework and/or CDR residues are mutated to a corresponding residue in a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived).

Additionally, the antibodies of the invention may contain any combination of two or more germline mutations within the framework and/or CDR regions, for example, wherein certain distinct residues are mutated to corresponding residues in a particular germline sequence. On the other hand, certain other residues that differ from the original germline sequence are retained or mutated to corresponding residues in the different germline sequence. Once obtained, antibodies containing one or more germline mutations may exhibit one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), or reduced It can be easily tested for immunogenicity, etc. Antibodies obtained by these general methods are included within the present invention.

The invention also includes CD3 antibodies comprising variants of any of the VH, VL, and/or CDR amino acid sequences disclosed herein with one or more conservative substitutions. For example, the present invention provides conservative amino acids, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acids compared to any of the VH, VL, and/or CDR amino acid sequences disclosed in Table 1 herein. Includes CD3 antibodies having VH, VL, and/or CDR amino acid sequences with substitutions.

As used herein, the term "complex" or "complexed" refers to the association of two or more molecules that interact with each other through bonds and/or forces other than peptide bonds (e.g., van der Waals, hydrophobic, hydrophilic forces). refers to In one embodiment, the complex is a heteromultimer. As used herein, the term “protein complex” or “polypeptide complex” refers to a complex with a non-protein entity (including, but not limited to, a chemical molecule such as a toxin or a detection reagent) conjugated to a protein in a protein complex. What it includes must be understood.

As used herein, “CD3 antibody” refers to monovalent antibodies with single specificity, as well as “bispecific antibodies,” “bi-specific antibodies,” trispecific antibodies,” “bifunctional antibodies,” and “heteromultimers.” , “heteromultimeric complex,” “bispecific heterodimeric diabody,” “heteromultimeric polypeptide,” or bispecific heterodimeric IgG. In some embodiments of the invention, the invention CD3 antibodies are human antibodies or humanized antibodies.

As used herein, a “bispecific antibody” is a molecule comprising at least a first polypeptide and a second polypeptide, wherein the second polypeptide differs in amino acid sequence from the first polypeptide by one or more amino acid residues. In some cases, bispecific antibodies are artificial hybrid antibodies that have two different heavy and light chain regions. Preferably, bispecific antibodies have binding specificity for two or more different ligands, antigens or binding sites. Therefore, a bispecific antibody can bind two different antigens simultaneously. The two antigen binding sites of a bispecific antibody bind to two different epitopes present on the same or different protein targets, for example tumor targets.

As used herein, the first and second polypeptide chains of a “bispecific antibody” comprise at least one antibody VL and one antibody VH region, or fragments thereof, wherein both antibody binding domains are within a single polypeptide chain. Included, the VL and VH regions of each polypeptide chain are from different antibodies.

Bispecific antibodies may be derived from full-length antibodies or antibody fragments (eg, F(ab') ₂ bispecific antibodies). Diabodies include, for example, EP404,097; WO93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993]. Bispecific antibodies are heterodimers of two “crossover” sFv fragments in which the VH and VL regions of the two antibodies are on different polypeptide chains.

As used herein, the terms “linked,” “fused,” “fused,” “covalently linked,” “covalently coupled,” and “genetically fused” are used interchangeably. These terms refer to joining two elements or components together by any means, including chemical conjugation or recombinant means. As used herein, the term “covalently linked” means that the specified moieties are covalently linked to one another either directly or indirectly through intervening moieties, such as linking peptides or moieties. In a preferred embodiment, the moieties are covalently fused. One type of covalent linkage is a peptide bond. Chemical conjugation methods (eg using heterobifunctional crosslinking agents) are known in the art. Additionally, fused moieties can be genetically fused. As used herein, the terms “genetically fused,” “genetically linked,” or “gene fused” refer to two or more proteins via individual peptide backbones by gene expression of a single polynucleotide molecule encoding a protein, polypeptide, or fragment. , refers to a covalent linkage or attachment of a polypeptide or fragment thereof. These gene fusions result in the expression of a single contiguous gene sequence. Preferred gene fusions are in frame, that is, two or more open reading frames (ORFs) are fused in a way that maintains the exact reading frame of the original ORF to form a continuous longer ORF. Accordingly, the recombinant fusion protein produced is a single polypeptide containing two or more protein segments corresponding to the polypeptide encoded by the original ORF (segments are generally not so linked in nature). In this case, a single polypeptide is cleaved during processing to produce a dimeric molecule containing two polypeptide chains.

As used herein, the term "linked" or "connected" refers to a direct peptide bond linkage between a first amino acid sequence and a second amino acid sequence, or to a first and a second amino acid sequence and between a first amino acid sequence and a second amino acid sequence. refers to a linkage comprising a third amino acid sequence peptide linked to. For example, a linker peptide is linked to the C-terminal end of one amino acid sequence and the N-terminal end of the remaining amino acid sequence.

As used herein, the term “linker” refers to an amino acid sequence that is two or more amino acids in length. Linkers can be made of neutral polar or non-polar amino acids. The linker can be, for example, 2 to 100 amino acids in length, such as 2 to 50 amino acids in length, for example 3, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids in length. You can have Linkers may be “cleavable” by autocleavage, or by cleavage by enzymes or chemicals. The cleavage sites of amino acid sequences, enzymes, and chemicals are well known in the art and are described herein.

As used herein, the term “disulfide bond” or “cysteine-cysteine disulfide bond” refers to a covalent interaction between two cysteines in which the sulfur atom of the cysteine is oxidized to form a disulfide bond. The average bond energy for disulfide bonds is about 60 kcal/mol, compared to 1 to 2 kcal/mol for hydrogen bonds. In the present invention, cysteine forming a disulfide bond is present in the framework region of a single chain antibody and serves to stabilize the shape of the antibody. Cysteine residues can be introduced, for example, by site-directed mutagenesis to create stabilizing disulfide bonds within the molecule.

The “knob-in-hole designation” is similar to the “protrusion and cavity” designation and can be used interchangeably.

“Protuberances” or “knobs” protrude from the contacts of the first polypeptide and thus the adjacent contacts (i.e. the contacts of the second polypeptide) to stabilize the heterodimer and thereby encourage heterodimer formation rather than homodimer formation. refers to one or more amino acid side chains that can be positioned in complementary cavities. The protuberances may be present in the original contact point or may be introduced synthetically (e.g., by altering the nucleic acid encoding the contact point). Typically, the nucleic acid encoding the contact point of the first polypeptide is altered to encode the protuberance. To achieve this, nucleic acids encoding one or more “original” amino acid residues at the junction of the first polypeptide are replaced with nucleic acids encoding one or more “imported” amino acid residues having a side chain volume greater than that of the original amino acid residues. The upper limit on the number of original residues replaced is the total number of residues in the contacts of the first polypeptide. Specific import residues for formation of protrusions are generally naturally occurring amino acid residues and are preferably selected from arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W).

A protrusion or knob is "positionable" in a cavity or hole, meaning the spatial location of the protrusion and cavity on the contact point of the first polypeptide and the second polypeptide, respectively, and the size of the protrusion and cavity is such that the protrusion is positioned at the contact point on the first and second polypeptides. This is to enable it to be located in the cavity without significantly perturbing its normal assembly. Because protrusions, such as phenylalanine (F), tyrosine (Y) and tryptophan (W), typically do not extend perpendicularly from the axis of the contact, the alignment of protrusions and corresponding cavities can be determined using, for example, X-ray crystallography or nuclear magnetic resonance (NMR). It relies on the modeling of protrusion/cavity pairs based on the three-dimensional structure obtained in . This can be accomplished using techniques widely recognized in the art.

A “cavity” or “hole” refers to one or more amino acid side chains that form a recess from the contact point of a second polypeptide and thus accommodate a corresponding protuberance on the contact point of an adjacent first polypeptide. The cavity may be present in the original contact point, or may be introduced synthetically (e.g. by altering the nucleic acid encoding the contact point). Typically, the nucleic acid encoding the contact point of the second polypeptide is altered to encode the cavity. To achieve this, the nucleic acid encoding one or more “original” amino acid residues of the contact point of the second polypeptide is replaced with DNA encoding one or more “import” amino acid residues having a smaller side chain volume than the original amino acid residue. The upper limit on the number of original residues replaced is the total number of residues in the contacts of the second polypeptide. Specific import residues for cavity formation are generally naturally occurring amino acid residues and are preferably selected from alanine (A), serine (S), threonine (T) and valine (V).

As used herein, “target antigen”, “target cell antigen”, “tumor antigen” or “tumor-specific antigen” refers to an antigen presented on the surface of a target cell, e.g., a cell within a tumor, such as a cancer cell or a cell of the tumor stroma. Refers to an antigenic determinant.

As used herein, the term “cancer” or “cancerous” refers to a physiological disease in mammals typically characterized by uncontrolled cell growth, neoplasm or tumor, resulting from abnormal and uncontrolled cell growth, or describe. In some aspects, cancer refers to a malignant primary tumor without localized metastases. In another aspect, cancer refers to a malignant tumor that invades or destroys neighboring body structures and spreads to distant sites. In some aspects, the cancer is associated with a specific cancer antigen. Examples of cancer include, but are not limited to, cancers of the oral cavity and pharynx, cancers of the digestive system (e.g., esophagus, stomach, small intestine, colon, rectum, liver, gallbladder, bile ducts, and pancreas), and cancers of the respiratory system (e.g., larynx, lungs, and bronchial tubes). (including non-small cell lung carcinoma), bone and joint cancers (e.g., bone metastases), soft tissue cancers, skin cancers (e.g., melanoma), breast cancer, reproductive system (e.g., cervix, corpus, ovaries, vulva, vagina) , prostate and testicles), urinary system (e.g. bladder, kidney, renal pelvis and ureter) cancer, eye and orbital cancer, brain cancer and nervous system cancer (e.g. glioma), head and neck cancer, or endocrine system (e.g. thyroid). ) includes cancer. Additionally, the cancer may be lymphoma (e.g., Hodgkin's disease and non-Hodgkin's lymphoma), multiple sclerosis, or leukemia (e.g., acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, etc.). In certain embodiments, the cancer of the digestive system is esophageal cancer, stomach cancer, small intestine cancer, colon cancer, rectal cancer, anal cancer, liver cancer, gallbladder cancer, appendix cancer, bile duct cancer, and pancreatic cancer.

With respect to cancer, the treatment is useful for any one or more of the following: (a) treating or preventing one or more symptoms of a disease associated with malignant cells (e.g., cancer of gastrointestinal involvement, such as colorectal cancer (CRC)) in an individual; do or improve; (b) inhibiting tumor growth or progression in an individual with a malignant tumor expressing tumor antigens; (c) inhibiting metastasis of cancer (malignant) cells expressing tumor antigens in an individual having one or more malignant cells expressing tumor antigens; (d) inducing regression (e.g., organ regression) of tumors expressing tumor antigens; (e) exerting cytotoxic activity on malignant cells expressing tumor antigens; (f) increasing progression-free survival in individuals with tumor antigen-related disorders; (g) increasing overall survival of individuals with tumor antigen-related disorders; (h) reducing the use of additional chemotherapeutic or cytotoxic agents in individuals with tumor antigen-related disorders; (i) reducing tumor burden in individuals with tumor antigen-related disorders; or (j) blocking the interaction of unidentified factors with tumor antigens. Without wishing to be bound by theory, treatment causes inhibition, ablation or death of cells in vitro or in vivo, or reduces the ability of cells, including mutated cells, to mediate a disorder, e.g., a disorder described herein, such as cancer. It is thought that

As used herein, the term “malignant cell” or “malignant tumor” refers to a tumor or tumor cells, i.e., cancerous cells, that are invasive and/or capable of undergoing metastasis.

Although any materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred materials and methods are described.

Materials and Methods

Conventional hybridoma methods for the production of monoclonal antibodies, recombinant techniques for the production of antibodies (including chimeric antibodies, e.g., humanized antibodies), antibody production in transgenic animals, and "fully human" antibodies. A variety of antibody production technologies have been described, including the recently described phage display technology for production. These techniques are briefly described below.

Polyclonal antibodies to an antigen of interest can generally be produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the antigen and adjuvant. The antigen (or fragment containing the target amino acid sequence) can be conjugated with a bifunctional or derivatizing agent, such as maleimidobenzoyl sulfosuccinimide ester (conjugated through a cysteine residue) or N-hydroxysuccinimide (via a lysine residue). ) can be used to conjugate a protein that is immunogenic in the species to be immunized, such as serum albumin, bovine thyroglobulin or soybean trypsin inhibitor. Animals are immunized against the immunogenic conjugate or derivative, and several weeks later the animals are boosted by subcutaneous injections at multiple sites. After 7 to 14 days, animals are bled and sera are analyzed for antibody titers. Animals are boosted to titer plateau. Preferably, the animal is boosted with a conjugate of the antigen conjugated to the same but different protein and/or via a different cross-linking reagent. Conjugates can also be produced in recombinant cell culture as protein fusions. Additionally, coagulants such as alum are used to enhance the immune response.

Monoclonal antibodies can be obtained from a population of substantially homogeneous antibodies using the hybridoma method first described by Kohler & Milstein, Nature 256:495, 1975, or by recombinant DNA methods (US 4,816,567 (Cabilly et al.) ) can be produced by. In the hybridoma method, a mouse or other suitable host animal is immunized as described above to induce lymphocytes that produce or are capable of producing antibodies capable of specifically binding to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusion agent, such as polyethylene glycol, to produce hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)) The hybridoma cells thus produced are seeded and grown in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. Additionally, the hybridoma cells may be used to produce ascites in the animal. Can grow in vivo as a tumor.Monoclonal antibodies secreted by subclones are purified from the culture medium by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography. , is suitably separated from ascites fluid or serum.

Expression of the antibody, antigen-binding fragment of the antibody, or any antibody construct is performed in a suitable prokaryotic or eukaryotic host cell, such as CHO cells, NSO cells, SP2/0 cells, HEK293 cells, COS cells, PER.C6 cells, yeast or Escherichia It can be performed in E. coli cells and the secreted antibodies are recovered and harvested from the cells (cell culture supernatant, conditioned cell culture supernatant, cell lysate or purified bulk). General antibody production methods are well known in the art and are described, for example, in Makrides, S.C., Protein Expr. Purif. 17:183-202, 1999]; [Geisse, S., et al., Protein Expr. Purif. 8:271-282, 1986]; [Kaufman, R.J., MoI. Biotechnology. 16:151-160, 2000]; [Werner, R.G., Drug Res. 48:870-880, 1998]. In a specific embodiment, the cell culture is a mammalian cell culture, such as Chinese hamster ovary (CHO) cell culture.

In one embodiment, the antibody may be recovered or isolated from a naturally producing source. In some such embodiments, the isolated or recovered antibody may be subjected to additional purification steps using conventional chromatographic methods known in the art. In particular, purification methods include, but are not limited to, affinity chromatography (e.g., Protein A affinity chromatography), ion exchange chromatography (e.g., anion exchange chromatography or cation exchange chromatography), hydrophobic interaction chromatography, hydrophobic interaction chromatography, hydrophobic interaction chromatography, and hydrophobic interaction chromatography. It is believed to include roxiapatite chromatography, gel filtration chromatography and/or dialysis. Among these, a preferred purification method is to use Protein A chromatography. The matrix to which the affinity ligand is attached is most often agarose, but other matrices can be used.

Other antibody purification techniques, such as ion exchange column fractionation, ethanol precipitation, reversed phase high pressure chromatography, ethanol precipitation, reversed phase HPLC, silica chromatography, Heparin Sepharose™ chromatography, anion or cation exchange resin chromatography (e.g. polyaspartic acid columns), chromatofocusing, electrophoresis using SDS-PAGE, and ammonium sulfate precipitation are also known in the art. The above list of purification methods is illustrative only and is not intended to be a limiting listing.

Alternatively, it is possible to produce transgenic animals (e.g. mice) that can produce the full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that homozygous deletion of the antibody heavy chain binding region (J.sub.H) in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene array in these germline mutant mice will result in the production of human antibodies upon antigenic challenge. See, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551-255;1993 and Jakobovits et al., Nature 362:255-258, 1993.

In some embodiments, antibodies of the invention can be humanized while maintaining high affinity for the antigen and other advantageous biological properties. Methods for humanizing non-human antibodies are well known in the art. Humanization can be performed essentially according to the method of Winter and colleagues (Jones et al., Nature 321:522-525, 1986) by substituting rodent CDRs or CDR sequences with the corresponding human antibody sequences. [Riechmann et al., Nature 332:323-327, 1988]; [Verhoeyen et al., Science 239:1534-1536, 1988]). Accordingly, these humanized antibodies are substantially chimeric antibodies (Cabilly, supra) in which less intact human variable domains are replaced by corresponding sequences from non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues, and possibly some FR residues, have been replaced by residues from similar regions in rodent antibodies. It is important to humanize antibodies while maintaining high affinity for the antigen and other advantageous biological properties. To achieve this goal, according to a preferred method, humanized antibodies are produced by an analysis process of the parent sequence and various conceptual humanized products using three-dimensional models of the parent sequence and the humanized sequence. Three-dimensional immunoglobulin models are familiar to those skilled in the art. Computer programs are available that display and display the probable three-dimensional conformational structure of selected candidate immunoglobulin sequences. Review of these displays allows analysis of the expected role of the residues in the function of the candidate immunoglobulin sequence, that is, analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected from consensus and import sequences and combined to achieve the desired antibody characteristics, such as increased affinity for the target antigen. For further details see WO 92/22653 (published on 23 December 1992).

Additionally, the antibodies of the present invention can be produced using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles carrying the polynucleotide sequence encoding them. In certain aspects, such phages can be used to display antigen binding domains, such as Fab and Fv, or disulfide-linked stabilized Fv, expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds to the antigen of interest can be selected or identified using the antigen, for example, a labeled antigen or an antigen bound or captured on a solid surface or bead. Phages used in these methods are typically filamentous phages including fd and M13. The antigen binding domain is expressed as a protein recombinantly fused to the phage gene III or gene VIII protein. Examples of phage display methods that can be used to produce immunoglobulins of the invention or fragments thereof are described in Brinkmann et al. “Phage Display Of Disulfide-Stabilized Fv Fragments,” J. Immunol. Methods, 182:41-50, 1995.

The functional characteristics of many IgG isotypes and their domains are well known in the art. The amino acid sequences of IgG1, IgG2, IgG3 and IgG4 are known in the art. The selection and/or combination of two domains from a specific IgG isotype for use in the methods of the invention can be based on any known parameters of the parent isotype, including affinity for FcyRs. For example, the use of regions or domains from an IgG isotype (e.g., IgG2 or IgG4) that may or may not exhibit limited binding to FcγRIIB may allow the bispecific antibody to maximize binding to activating receptors and to inhibit inhibitory receptors. It may have a specific application where it is intended to be manipulated to minimize binding to. Similarly, the use of Fc chains or domains from IgG isotypes known to preferentially bind to C1q or FcγRIIIA (e.g. IgG3) can be used to maximize effector function activity, e.g. complement activation or ADCC. Antibodies can be combined with Fc amino acid modifications known in the art to enhance antibody dependent cell mediated cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) to engineer antibodies. In a similar manner, the Fc chain or domain of an IgG isotype can be mutated to minimize or eliminate the effector functions of the Fc chain.

During the discovery process, production and characterization of antibodies can elucidate information about desirable epitopes. From this information, it is possible to competitively screen antibodies for binding to the same epitope. An approach to achieve this is to perform competition and cross-competition to find antibodies that compete or cross-compete with each other, e.g. antibodies that compete for binding to the antigen. One method is to identify the epitope to which the antibody binds, or "epitope mapping." Analysis of crystal structures of antibody-antigen complexes, competition assays, as described, for example, in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999. Many methods are known in the art for mapping and characterizing the location of epitopes on proteins, including gene fragment expression analysis and synthetic peptide-based analysis. In a further example, epitope mapping can be used to determine the sequence to which an antibody binds. Epitope mapping is commercially available from various sources, such as Pepscan Systems (Edelhertweg 15, 8219 PH Lelystad, The Netherlands). In additional examples, mutagenesis of the antigen binding domain, domain exchange experiments, and alanine scanning mutagenesis can be performed to identify residues that are necessary, sufficient, and/or essential for epitope binding. The binding affinity and dissociation rate of antigen binding domain interactions can be determined by competition binding assays. One example of a competitive binding assay is a radioimmunoassay that involves incubation of labeled antigen and detection of molecules bound to the labeled antigen. The affinity and binding dissociation rate of molecules of the invention for an antigen can be determined from saturation data by Scatchard analysis.

The affinity and binding properties of the antibodies of the invention for an antigen can be determined using an antigen-binding assay, including but not limited to enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) analysis, bio-layer interferometry, or immunoprecipitation assay. The binding domain can be initially determined using in vitro assays known in the art (biochemical or immunology based assays). Molecules of the invention may have similar binding properties in in vivo models (such as those described and disclosed herein) compared to those in in vitro based assays. However, the present invention excludes molecules of the invention that do not exhibit the desired phenotype in in vitro based assays but do exhibit the desired phenotype in vivo.

After obtaining the nucleic acid sequence encoding the molecule (i.e., binding domain) of the invention, vectors for production of the molecule can be produced by recombinant DNA technology using methods well known in the art.

A polynucleotide encoding an antibody binding domain of the invention may comprise an expression control polynucleotide sequence operably linked to an antibody coding sequence comprising a naturally associated or heterologous promoter region known in the art. The expression control sequence may be the eukaryotic promoter system of a vector capable of transforming or transfecting eukaryotic host cells, but control sequences for prokaryotic hosts may also be used. After the vector has been incorporated into an appropriate host cell line, the host cells are propagated under conditions suitable for expressing the nucleotide sequence and, if desired, for collection and purification of the antibody. Eukaryotic cell lines include CHO cell lines, various COS cell lines, HeLa cells, myeloma cell lines, transformed B-cells, or human embryonic kidney cell lines.

In one embodiment, the DNA encoding an antibody of the invention is cloned using routine procedures (e.g., by using oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of a murine antibody). Can be isolated and sequenced. Hybridoma cells of the invention serve as a preferred source of such DNA. After isolation, the DNA can be placed into an expression vector, which can then be transfected into host cells that do not otherwise produce immunoglobulin proteins, such as simian COS cells, CHO cells, or myeloma cells, to produce a composite of the monoclonal antibody in the recombinant host. obtain. DNA can also be modified, for example, by substituting the coding sequences for human heavy and light chain constant domains in place of the homologous murine sequences (Morrison et al., Proc. Nat. Acad. Sci. 81:6851, 1984]). In this way, chimeric antibodies are produced that have the binding specificity of the anti-antigen monoclonal antibodies herein.

CD3 antibody

In one aspect, the invention provides agents that specifically bind to CD3 (e.g., the full-length human CD3 epsilon subunit (e.g., Accession No. NP_000724 or SEQ ID No. 66)).

In certain embodiments, the CD3 antibody specifically binds human CD3.

Exemplary CD3 antibodies of the invention are listed herein in Tables 1, 2, and 3. Table 1 presents the amino acid sequence identifiers of the VH region and VL region. In some embodiments, antibodies are provided having any one of the partial light chain sequences listed in Table 1 and/or any one of the partial heavy chain sequences listed in Table 1.

[Table 1]

In Table 1, the underlined sequences are the CDR sequences according to Kabat and the bold letters are according to Chotia.

The present invention provides CDR portions of antibodies against CD3 (including the Chotia, Kabat CDRs, and CDR contact regions). Methods and techniques for identifying CDRs within VH and VL amino acid sequences are well known in the art and can be used to identify CDRs within a specified VH and/or VL amino acid sequence disclosed herein. Exemplary conventions that can be used to identify the boundaries of a CDR include, for example, the Kabat definition, Chotia definition, and AbM definition. Broadly speaking, the Kabat definition is based on sequence variability, the Chotia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chotia approaches. See, for example, Kabat, “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1991 )]; [Al-Lazikani et ai, J. Mol. Biol. 273:927-948 (1997)]; and [Martin et ai, Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989).

Public databases are also available for identifying CDR sequences in antibodies. It is understood that, in some embodiments, a CDR may be a combination of Kabat and Chotia CDRs (also referred to as “combined CR” or “extended CDR”). In some embodiments, the CDR is a Kabat CDR. In some additional embodiments, the CDR is a Chotia CDR. In other words, for embodiments with more than one CDR, the CDRs can be any of Kabat, Chotia, combination CDRs, or combinations thereof. Tables 2 and 3 provide examples of CDR sequences provided herein.

In one embodiment, the invention provides an antibody comprising an amino acid sequence listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

In some such embodiments, the invention provides an amino acid sequence of any of the VL region amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. It provides an antibody comprising a VL region comprising a.

In some further such embodiments, the invention provides an amino acid sequence of any of the VH region amino acid sequences listed in Table 1, or substantially at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. Antibodies comprising a VH region comprising similar sequences are provided.

In one embodiment, an antibody is provided that specifically binds to CD3, wherein the antibody comprises: (a) determining the VH complement of the VH sequence shown in SEQ ID NO: 2, 4, 5, 7, 10, 12 or 35 a heavy chain variable (VH) region comprising region 1 (VH CDR1), VH complement determining region 2 (VH CDR2), and VH complement determining region 3 (VH CDR3); and/or (b) VL complementation determining region 1 (VL CDR1), VL complementation determining region 2 (VL CDR2) of the VL sequence shown in SEQ ID NO: 1, 3, 6, 8, 9, 11, 34, 87 or 89, and Light chain variable (VL) region containing VL complementation determining region 3 (VL CDR3).

In one embodiment, the invention provides a set of six CDRs (i.e. VL CDR1, VL CDR2, VL CDR3, VH CDR1, Provides an antibody comprising VH CDR2, VH CDR3).

In specific embodiments, the invention provides SEQ ID NOs: 1 and 2 (e.g. 2B5-0001(2B5)); SEQ ID NO: 3 and 2 (2B5-0002); SEQ ID NOs: 3 and 4 (e.g. 2B5-0006 (2B5v6)); SEQ ID NOs: 3 and 5 (e.g. 2B5-0009); SEQ ID NOs: 6 and 7 (e.g. 2B5-0517); SEQ ID NOs: 8 and 7 (e.g. 2B5-0522); SEQ ID NOs: 6 and 4 (e.g. 2B5-0533); SEQ ID NOs: 8 and 4 (e.g. 2B5-0538); SEQ ID NOs: 9 and 10 (e.g. 2B5-0598); SEQ ID NOs: 9 and 7 (e.g. 2B5-0623); SEQ ID NOs: 11 and 12 (eg 2B5-0707); SEQ ID NOs: 34 and 35 (eg 2B5-0003); SEQ ID NOs: 85 and 2 (eg H2B4); SEQ ID NOs: 9 and 4 (e.g. 2B5-1038); SEQ ID NOs: 87 and 4 (e.g. 2B5-1039); And a set of VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2 or VH CDR3 amino acid sequences contained within a VL / VH amino acid sequence pair selected from the group consisting of SEQ ID NOs: 89 and 4 (e.g. 2B5-1040). Provides antibodies that

Table 2 provides the VH CDR1, VH CDR2, and VH CDR3 regions of exemplary CD3 antibodies.

[Table 2]

In one embodiment, the invention includes an amino acid sequence of any of the VH CDR1 amino acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. Provided is an antibody comprising VH CDR1.

In another embodiment, the invention provides an amino acid sequence of any of the VH CDR2 amino acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. Provided is an antibody comprising a VH CDR2 comprising the antibody.

In another embodiment, the invention provides an amino acid sequence of any of the VH CDR3 amino acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. An antibody comprising a VH CDR3 is provided.

Table 3 provides the VL CDR1, VL CDR2, and VL CDR3 of exemplary CD3 antibodies.

[Table 3]

In one embodiment, the invention includes an amino acid sequence of any of the VL CDR1 amino acid sequences listed in Table 3, or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. Provided is an antibody comprising the VL CDR1.

In another embodiment, the invention provides an amino acid sequence of any of the VL CDR2 amino acid sequences listed in Table 3, or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. Provided is an antibody comprising a VL CDR2 comprising the antibody.

In another embodiment, the invention provides an amino acid sequence of any of the VL CDR3 amino acid sequences listed in Table 3, or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. An antibody comprising a VL CDR3 is provided.

In some embodiments, an antibody is provided that specifically binds to CD3, wherein the antibody comprises: (a) an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 5, 7, 10, 12, and 35. a heavy chain (HC) variable (VH) region comprising the VH complement determining region 1 (CDR1), VH complement determining region 2 (VH CDR2), and VH complement determining region 3 (VH CDR3) of the VH; and/or (b) VL complementary determining region 1 (VL CDR1), VL complementary determining region of a VL having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 6, 8, 9, 11, 34, 87 and 89. Light chain (LC) variable (VL) region comprising VL complementation determining region 2 (VL CDR2) and VL complementation determining region 3 (VL CDR3).

In a specific embodiment, the antibody comprises: (a) (i) a VH CDR1 comprising the sequence of SEQ ID NO: 13, 14, or 15; (ii) a VH CDR2 comprising the sequence of SEQ ID NO: 16, 17, 19, 20, 21, 22, 23 or 24; and (iii) a VH region comprising a VH CDR3 comprising the sequence of SEQ ID NO: 18 or 25; and/or (b) (i) a VL CDR1 comprising the sequence of SEQ ID NO: 26, 29, 30, 32, 91 or 92; (ii) VL CDR2 comprising the sequence of SEQ ID NO: 27 or 31; and (iii) a VL region comprising a VL CDR3 comprising the sequence of SEQ ID NO: 28 or 33.

In certain embodiments, the invention provides a , 2B5-0623, 2B5-0707, 2B5-0003, 2B5-1038, 2B5-1039, and 2B5-1040.

In some embodiments, the invention also provides CDR portions of CDR antibodies based on CDR contact regions. The CDR contact region is a region of the antibody that confers specificity to the antibody for the antigen. Typically, the CDR contact region includes residue positions in the CDR and Vernier bands that are restricted to maintain an appropriate loop structure for the antibody to bind to the specific antigen. See, for example, Makabe et al., J. Biol. Chem., 283:1156-1166, 2007. Determination of the CDR contact area is within the skill of the art.

Particular CD3 antibodies provided herein may be referred to by more than one name. For example, 2B5-0001 may be referred to as 2B5 or h2B5; 2B5-0006 may be referred to as 2B5v6 or h2B5v6. Table 4 shows the format of the antibodies and the CD3 antibody regions present in the antibodies disclosed herein.

[Table 4]

The binding affinity (K _D ) of the antibodies described herein to CD3 (e.g., human CD3 epsilon (e.g., SEQ ID NO:40)) may be from about 0.001 to about 6500 nM. In some embodiments, the binding affinity is about 6500 nM, 6000 nM, 5500 nM, 4500 nM, 4000 nM, 3500 nM, 3000 nM, 2500 nM, 2000 nM, 1500 nM, 1000 nM, 750 nM, 500 nM, 400 nM. nM, 300 nM, 250 nM, 200 nM, 150 nM, 100 nM, 75 nM, 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 19 nM, 17 nM, 16 nM, 15 nM, 10 nM, 8 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5.5 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.3 nM, 0.1 nM, 0.01 nM , 0.002 nM or 0.001 nM. In some embodiments, the binding affinity is about 6500 nM, 6000 nM, 5500 nM, 5000 nM, 4000 nM, 3000 nM, 2000 nM, 1000 nM, 900 nM, 800 nM, 500 nM, 250 nM, 200 nM, 100 nM. nM, 50 nM, 30 nM, 20 nM, 10 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5 nM, 4.5 nM, 4 nM, 3.5 nM, 3 nM, 2.5 nM, 2 nM, 1.5 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM or less nM. In certain embodiments, the antibody has a K _D of about 80 to about 200 pM. In certain embodiments, the antibody has a K _D of about 10 to 2000 nM.

In one aspect, the present invention provides an antibody or pharmaceutical composition for use in a method of modulating a T cell-mediated immune response in an individual in need of such modulation. In specific embodiments, the invention provides antibodies or pharmaceutical compositions for use in a method of inhibiting the growth of tumor cells in an individual.

In another aspect, the present invention provides an antibody or pharmaceutical composition for use in the treatment of cancer, optionally comprising breast cancer, ovarian cancer, thyroid cancer, prostate cancer, uterine cancer, lung cancer, bladder cancer, endometrial cancer, head and neck cancer, It is selected from the group consisting of testicular cancer, glioblastoma and cancer of the digestive system.

In another aspect, the invention provides an antibody or pharmaceutical composition for use in the treatment of cancer, wherein a T cell mediated immune response is modulated or the growth of tumor cells is inhibited.

Additionally, the present invention provides nucleic acid molecules encoding CD3 antibodies or portions thereof. For example, the invention provides a nucleic acid molecule encoding any of the VH or VL amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. provides.

The invention also provides for any VH CDR1, VH CDR2 or VH CDR3 amino acid sequence listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. Provides a nucleic acid molecule that

Additionally, the present invention provides any VL CDR1, VL CDR2 or VL CDR3 amino acid sequence listed in Table 3, or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. Provides an encoding nucleic acid molecule.

The invention also provides a nucleic acid molecule encoding a HC VR, wherein the VH comprises a set of three CDRs (i.e. VH CDR1, VH CDR2 or VH CDR3), and the VH CDR1, VH CDR2 and VH CDR3 amino acid sequence sets are shown in Table As defined by any of the exemplary CD3 antibodies listed in 2.

The invention also provides a nucleic acid molecule encoding an LC VR, wherein the VL comprises a set of three CDRs (i.e. VL CDR1, VL CDR2 or VL CDR3), and the VL CDR1, VL CDR2 and VL CDR3 amino acid sequence sets are shown in Table As defined by any of the exemplary CD3 antibodies listed in 3.

The invention also provides nucleic acid molecules encoding both a VH and a VL, wherein the VH comprises an amino acid sequence of any of the VH amino acid sequences listed in Table 1, and the VL comprises an amino acid sequence of any of the VL amino acid sequences listed in Table 1. Includes.

Additionally, the present invention provides a recombinant expression vector capable of expressing a polypeptide comprising the heavy or light chain variable region of a CD3 antibody. For example, the invention includes recombinant expression vectors comprising any of the nucleic acid molecules described above, i.e., nucleic acid molecules encoding any of the VH and VL sequences set forth in Table 1 and/or the CDR sequences set forth in Tables 2 or 3. Additionally, host cells into which these vectors are introduced; and culturing said host cells under conditions permissive for producing antibodies or antibody fragments, and recovering the antibodies and antibody fragments thus produced. Methods for producing antibodies or portions thereof are within the scope of the present invention. belongs

In certain embodiments, the polynucleotide is an antibody 2B5-0001 (2B5), 2B5-0002, 2B5-0006 (2B5v6), 2B5-0009, 2B5-0517, 2B5-0522, 2B5-0533, 2B5-0538 listed in Table 4. , 2B5-0598, 2B5-0623, 2B5-0707, 2B5-0003, 2B5-1038, 2B5-1039 or 2B5-1040.

The sequence encoding the antibody of interest is preserved in the vector in the host cell, and the host cells can be expanded and frozen for future use. Vectors (including expression vectors) and host cells are further described herein.

Also encompassed by the invention are polynucleotides complementary to any of these sequences. In one aspect, the invention provides methods for producing any of the polynucleotides described herein. Polynucleotides can be single-stranded (coated or antisense) or double-stranded, and can be DNA (genomic, cDNA, or synthetic) or RNA molecules. RNA molecules include HnRNA molecules (which contain introns and correspond in a one-to-one manner to DNA molecules) and mRNA molecules (which do not contain introns). Additional coding or non-coding sequences may, but need not, be present within the polynucleotides of the invention, and the polynucleotides may, but need not, be linked to other molecules and/or support materials.

A polynucleotide may comprise a native sequence (i.e., an endogenous sequence encoding an antibody or portion thereof) or may comprise variants of such sequence. Polynucleotide variants contain one or more substitutions, additions, deletions and/or insertions such that the immunoreactivity of the encoded polypeptide is not attenuated compared to the native immunoreactive molecule. The effect of the encoded polypeptide on immune reactivity can be assessed generally as described herein. The variant preferably has at least about 70% identity, more preferably, at least about 80% identity, even more preferably, at least about 90% identity, and most preferably, to the polynucleotide sequence encoding the native antibody or portion thereof. represents at least about 95% identity.

The polynucleotide of the present invention can be obtained using chemical synthesis, recombinant methods, or PCR. Methods for chemical polynucleotide synthesis are well known in the art and need not be described in detail herein. One skilled in the art can produce the desired DNA sequence using the sequences provided herein and commercially available DNA synthesis equipment.

When recombinant methods are used to produce polynucleotides, polynucleotides containing the desired sequence can be inserted into a suitable vector, and the vector can then be transferred into a host cell suitable for replication and amplification, as further discussed herein. It can be introduced. Polynucleotides can be inserted into host cells by any means known in the art. Cells are transformed by introducing exogenous polynucleotides by direct uptake, endocytosis, transfection, F-matching, or electroporation. Once introduced, the exogenous polynucleotide may be maintained within the cell as a non-integrating vector (e.g., a plasmid) or may be integrated within the host cell genome. Polynucleotides thus amplified can be isolated from host cells by methods well known in the art (see, for example, Sambrook et al., 1989).

Alternatively, PCR allows reproduction of DNA sequences. PCR technology is well known in the art and is described in US 4,683,195, 4,800,159, 4,754,065 and 4,683,202 as well as PCR: The Polymerase Chain Reaction, Mullis et al. eds., Birkauswer Press, Boston, 1994.

RNA can be obtained by using isolated DNA in an appropriate vector and inserting it into a suitable host cell. Once the cell has been replicated and the DNA has been transcribed into RNA, the RNA can be isolated using methods well known to those skilled in the art, for example, as set forth in Sambrook et al., 1989, supra.

Suitable cloning vectors can be constructed according to standard techniques or can be selected from the numerous cloning vectors available in the art. The cloning vector selected may depend on the host cell to be used, but useful cloning vectors will generally have self-replicating capacity, may possess a single target for a particular restriction endonuclease, and/or contain a vector may contain genes for markers that can be used to select clones. Suitable examples include plasmids and bacterial viruses, such as pUC18, pUC19, Bluescript (e.g. pBS SK+) and derivatives thereof, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle. Vectors such as pSA3 and pAT28. These and many other cloning vectors are available from commercial sources such as BioRad, Strategene and Invitrogen.

Expression vectors are generally replicable polynucleotide constructs containing polynucleotides according to the invention. This means that the expression vector must be replicable within the host cell, either as an episome or as an integrated part of the chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors such as adenoviruses, adeno-associated viruses, retroviruses, cosmids, and the expression vectors disclosed in WO 87/04462. Vector components generally include, but are not limited to, one or more of the following: a signal sequence; origin of replication; one or more marker genes; Suitable transcription control elements (such as promoters, enhancers and terminators). For expression (i.e. translation), one or more translation control elements are typically also required, such as a ribosome binding site, translation initiation site, and stop codon.

Vectors containing the corresponding polynucleotide can be generated by electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; and infection (e.g., when the vector is an infectious agent such as vaccinia virus). The choice of introduction vector or polynucleotide will often depend on the characteristics of the host cell.

The invention also provides host cells comprising any of the polynucleotides described herein. Any host cell capable of overexpressing heterologous DNA can be used for the purpose of isolating the gene encoding the antibody, polypeptide, or protein of interest. Non-limiting examples of mammalian host cells include, but are not limited to, COS, HeLa, and CHO cells. See also WO 87/04462. Suitable non-mammalian host cells include prokaryotes (such as Escherichia coli or Bacillus subtilis ) and yeast (such as Saccharomyces cerevisiae , Schizosaccharomyces pombe ). ) or Kluyveromyces lactis ). Preferably, the host cell expresses the cDNA at a level about 5-fold, more preferably, 10-fold, even more preferably 20-fold greater than the corresponding endogenous antibody or protein CD3, or has a CD3 domain ( For example domains 1 to 4) are obtained by immunoassay or FACS. Cells overexpressing the antibody or protein of interest can be identified.

The present invention also includes scFvs of the antibodies of the present invention. Single chain variable region fragments are produced by linking light and/or heavy chain variable regions using short linking peptides (Bird et al., Science 242:423-426, 1988). Examples of linking peptides include the sequence of SEQ ID NO:65. In one embodiment, the linking peptide bridges about 3.5 nm between the carboxy terminus of one variable region and the amino terminus of the other variable region. Linkers of different sequences have been designed and used (Bird et al., Science 242:423-426, 1988). The linker should be a short, flexible polypeptide and preferably consist of less than about 20 amino acid residues. Linkers can be modified for additional functions, such as attachment of drugs or attachment to a solid support. Single chain variants can be produced recombinantly or synthetically. For synthetic production of scFv, automated synthesis equipment can be used. For recombinant production of scFv, a suitable plasmid containing a polynucleotide encoding the scFv can be introduced into a suitable host cell (eukaryotic, such as yeast, plant, insect or mammalian cell, or prokaryotic, such as Escherichia coli). . Polynucleotides encoding the scFvs of interest can be produced by conventional manipulations, such as ligation of polynucleotides. The scFv produced can be isolated using standard protein purification techniques known in the art.

In one aspect, a polynucleotide sequence encoding any one of the partial light chain sequences and/or any one of the partial heavy chain sequences disclosed herein is provided. Exemplary CD3 polynucleotides encoding the VH and VL regions of antibodies of the invention are listed in Table 5.

[Table 5]

In one aspect, the invention provides compositions (e.g., pharmaceutical compositions) comprising any of the polynucleotides of the invention. In some embodiments, the composition may include any one of the polynucleotides shown in Table 5. In a specific embodiment, the composition comprises an expression vector comprising a polynucleotide encoding any of the antibodies described herein, such as one of the polynucleotides shown in Table 5.

Also encompassed by the invention are polynucleotides complementary to any of these sequences. Polynucleotides can be single-stranded (coated or antisense) or double-stranded, and can be DNA (genomic, cDNA, or synthetic) or RNA molecules. RNA molecules include mature and immature mRNAs, such as precursor mRNA (pre-mRNA) or heteronuclear mRNA (hnRNA) and mature mRNA. Additional coding or non-coding sequences may, but need not, be present within the polynucleotides of the invention, and the polynucleotides may, but need not, be linked to other molecules and/or support materials.

A polynucleotide may comprise a native sequence (i.e., an endogenous sequence encoding an antibody or portion thereof) or may comprise variants of such sequence. Polynucleotide variants contain one or more substitutions, additions, deletions and/or insertions such that the immunoreactivity of the encoded polypeptide is not attenuated compared to the native immunoreactive molecule. The effect of the encoded polypeptide on immune reactivity can be assessed generally as described herein. The variant preferably has at least about 70% identity, more preferably, at least about 80% identity, even more preferably, at least about 90% identity, and most preferably, to the polynucleotide sequence encoding the native antibody or portion thereof. represents at least about 95% identity.

Two polynucleotide or polypeptide sequences are said to be “identical” if the sequences of nucleotides or amino acids within the two sequences are identical when aligned to the greatest extent possible as described below. Typically, comparisons between two sequences are performed by comparing the sequences over a comparison window and identifying and comparing local regions of sequence similarity. As used herein, “comparison window” refers to a segment of at least about 20 contiguous positions, typically 30 to about 75, or 40 to about 50 contiguous positions, where the sequences optimally align the two sequences. It can then be compared to the same number of reference sequences at adjacent positions.

Optimal alignment of sequences for comparison was performed using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, WI, USA) using default parameters. )) can be used. Preferably, the "percentage sequence identity" is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence within the comparison window is the optimal alignment of the two sequences. It may contain additions or deletions (i.e. gaps) of up to 20%, typically 5 to 15%, or 10 to 12%, compared to the reference sequence for alignment (not including additions or deletions). The percentage determines the number of positions where the same nucleic acid base or amino acid residue is present in both sequences, yielding the number of matched positions, and dividing the number of matched positions by the total number of positions in the reference sequence (i.e. window size). It is calculated by dividing by and multiplying the result by 100 to calculate the percentage of sequence identity.

A variant may additionally or alternatively be substantially homologous to a native gene, or a portion thereof, or a complement. These polynucleotide variants can hybridize under moderately stringent conditions to naturally occurring DNA sequences (or complementary sequences) encoding the native antibody.

It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode polypeptides as described herein. Some of these polynucleotides possess minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. Additionally, alleles of genes comprising the polynucleotide sequences provided herein are included within the scope of the present invention. An allele is an endogenous gene that has been altered as a result of one or more mutations, such as deletions, additions and/or substitutions, of nucleotides. The resulting mRNA and proteins may, but need not, have altered structure or function. Alleles can be identified using standard techniques (such as hybridization, amplification and/or database sequence comparison).

In one aspect, the invention provides methods for producing any of the polynucleotides described herein. For example, polynucleotides of the present invention can be obtained using chemical synthesis, recombinant methods, or PCR. Methods for chemical polynucleotide synthesis are well known in the art and need not be described in detail herein. One skilled in the art can produce the desired DNA sequence using the sequences provided herein and commercially available DNA synthesis equipment.

When recombinant methods are used to produce polynucleotides, polynucleotides containing the desired sequence can be inserted into a suitable vector, and the vector can then be transferred into a host cell suitable for replication and amplification, as further discussed herein. It can be introduced. Polynucleotides can be inserted into host cells by any means known in the art. Cells are transformed by introducing exogenous polynucleotides by direct uptake, endocytosis, transfection, F-matching, or electroporation. Once introduced, the exogenous polynucleotide may be maintained within the cell as a non-integrating vector (e.g., a plasmid) or may be integrated within the host cell genome. Polynucleotides thus amplified can be isolated from host cells by methods well known in the art (see, for example, Sambrook et al., 1989).

Alternatively, PCR allows reproduction of DNA sequences. PCR technology is well known in the art and is described in US 4,683,195, 4,800,159, 4,754,065 and 4,683,202 as well as PCR: The Polymerase Chain Reaction, Mullis et al. eds., Birkauswer Press, Boston, 1994.

RNA can be obtained by using isolated DNA in an appropriate vector and inserting it into a suitable host cell. Once the cell has been replicated and the DNA has been transcribed into RNA, the RNA can be isolated using methods well known to those skilled in the art, for example, as set forth in Sambrook et al., 1989, supra.

Suitable cloning vectors can be constructed according to standard techniques or can be selected from the numerous cloning vectors available in the art. The cloning vector selected may depend on the host cell to be used, but useful cloning vectors will generally have self-replicating capacity, may possess a single target for a particular restriction endonuclease, and/or contain a vector may contain genes for markers that can be used to select clones. Suitable examples include plasmids and bacterial viruses, such as pUC18, pUC19, BlueScript (e.g. pBS SK+) and derivatives thereof, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors such as Includes pSA3 and pAT28. These and many other cloning vectors are available from commercial sources such as BioRad, Straightgene, and Invitrogen.

Expression vectors are generally replicable polynucleotide constructs containing polynucleotides according to the invention. This means that the expression vector must be replicable within the host cell, either as an episome or as an integrated part of the chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors such as adenoviruses, adeno-associated viruses, retroviruses, cosmids, and the expression vectors disclosed in WO 87/04462. Vector components generally include, but are not limited to, one or more of the following: a signal sequence; origin of replication; one or more marker genes; Suitable transcription control elements (such as promoters, enhancers and terminators). For expression (i.e. translation), one or more translation control elements are typically also required, such as a ribosome binding site, translation initiation site, and stop codon.

Vectors containing the corresponding polynucleotide can be generated by electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; Microprojectile impact; lipofection; and infection (e.g., when the vector is an infectious agent such as vaccinia virus). The choice of introduction vector or polynucleotide will often depend on the characteristics of the host cell.

Any host cell capable of overexpressing heterologous DNA can be used for the purpose of isolating the gene encoding the antibody, polypeptide, or protein of interest. Non-limiting examples of mammalian host cells include, but are not limited to, COS, HeLa and CHO cells (see also WO 87/04462). Suitable non-mammalian host cells include prokaryotes (such as Escherichia coli or Bacillus subtilis) and yeast (such as Saccharomyces cerevisiae, Schizosaccharomyces pombe or Kluyveromyces lactis). . Preferably, the host cell expresses the cDNA at a level about 5-fold, more preferably, 10-fold, even more preferably 20-fold greater than the corresponding endogenous antibody or protein CD3, or has a CD3 domain ( For example domains 1 to 4) are obtained by immunoassay or FACS. Cells overexpressing the antibody or protein of interest can be identified.

In one aspect, the CD3 antibody molecule will have an affinity for CD3 in the picomolar to micromolar affinity range, preferably in the picomolar to low nanomolar range, as measured, for example, by direct binding or competitive binding assays.

Bispecific antibodies can be produced using the sequences disclosed herein. Methods for producing bispecific antibodies are known in the art (see, for example, Suresh et al., Methods in Enzymology 121:210, 1986). Typically, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, with the two heavy chains having different specificities (Millstein and Cuello, Nature 305, 537-539, 1983 ]).

In another embodiment, the antibodies described herein comprise full-length human antibodies, wherein the antibody variable region of the heterodimeric protein binds specifically to an effector antigen (e.g., a CD3 antigen) located on a human immune effector cell. Can recruit the activity of human immune effector cells, and the second antibody variable region of the heterodimeric protein can specifically bind to the target antigen. In some embodiments, the human antibody has an IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the heterodimeric protein comprises an immunologically inactive Fc chain.

Human immune effector cells can be any of a variety of immune effector cells known in the art. For example, immune effector cells can be members of the human lymphoid cell lineage, including, but not limited to, T cells (e.g., cytotoxic T cells), B cells, and natural killer (NK) cells. Additionally, immune effector cells can be members of the human myeloid lineage, including, but not limited to, monocytes, neutrophil granulocytes, and dendritic cells. Such immune effector cells may have cytotoxic or apoptotic effects on target cells, or other desired effects upon activation by binding of effector antigens.

An effector antigen is an antigen (eg, a protein or polypeptide) expressed on human immune effector cells. Examples of effector antigens capable of binding heterodimeric proteins (e.g., heterodimeric antibodies or bispecific antibodies) include, but are not limited to, human CD3, CD16, NKG2D, NKp46, CD2, CD28, CD25, CD64, and CD89. Includes.

The target cell may be a cell that is native or foreign to the human. In a natural target cell, the cell can be transformed into a malignant cell or pathologically transformed (e.g. a natural target cell infected by a virus, Plasmodium or bacteria). In a foreign target cell, the cell is an invading pathogen, such as bacteria, Plasmodium or a virus.

In some embodiments, antibodies useful in the invention include Fab, Fab fragment, F(ab) ₂ fragment, Fv fragment, single chain Fv fragment, disulfide stabilized Fv fragment, single chain antibody, monoclonal antibody, chimeric antibody, bispecific antibody. It is an antibody, trispecific antibody, multispecific antibody, bispecific heterodimeric diabody, bispecific heterodimeric IgG, polyclonal antibody, labeled antibody, humanized antibody, human antibody, or fragment thereof. .

In some embodiments, the CD3 antibody described herein is a monoclonal antibody. For example, the CD3 antibody is a humanized monoclonal antibody or a chimeric monoclonal antibody.

The present invention includes antibodies, or portions thereof, comprising an Fc chain or domain. In some embodiments, the Fc chain or portion thereof comprises one or more constant domains (e.g., CH2 or CH3 domains) of an Fc chain of IgG1, IgG2, IgG3, or IgG4. In another embodiment, the invention includes a molecule comprising an Fc chain or portion thereof, wherein the Fc chain or portion thereof comprises one or more amino acid modifications (e.g., substitutions) compared to a similar wild-type Fc chain or portion thereof. do. Variant Fc regions are well known in the art and do not alter the phenotype of an antibody comprising the variant Fc region as analyzed in any binding activity or effector function assay well known in the art, such as ELISA, SPR analysis or ADCC. It is mainly used to Such variant Fc chains or portions thereof may extend the plasma half-life and stability exhibited by the bispecific antibodies of the invention comprising an Fc chain or portions thereof. In another embodiment, the invention encompasses the use of any Fc variant known in the art.

In one embodiment, one or more modifications occur to amino acids in the Fc chain to reduce the affinity and avidity of the Fc chain, and thus the bispecific antibody of the invention, for one or more FcγR receptors. In specific embodiments, the invention includes a bispecific antibody comprising a variant Fc chain or portion thereof, wherein the variant Fc chain is a wild-type Fc chain wherein the variant Fc region binds only one FcγR, wherein the FcγR is FcγRIIIA. Contains one or more amino acid modifications compared to In another specific embodiment, the invention includes a bispecific antibody comprising a variant Fc chain or portion thereof, wherein the variant Fc chain is a wild type antibody wherein the variant Fc region binds only one FcγR, wherein the FcγR is FcγRIIA. (WT) contains one or more amino acid modifications compared to the Fc chain. In another specific embodiment, the invention includes a bispecific antibody comprising a variant Fc chain or portion thereof, wherein the variant Fc chain is a wild type antibody that binds to only one FcγR, wherein the FcγR is FcγRIIB. Contains one or more amino acid modifications compared to the Fc chain. In another embodiment, the invention includes molecules comprising a variant Fc chain, wherein the variant does not comprise an Fc chain or comprises a wild-type Fc chain as measured using methods known to those skilled in the art or described herein. confers or mediates comparatively reduced ADCC activity (or other effector functions) and/or increased binding to FcγRIIB (CD32B).

The invention also includes the use of an Fc region comprising domains or regions of two or more IgG isotypes. As is known in the art, amino acid modifications of the Fc region can profoundly affect Fc-mediated effector function and/or binding activity. However, these changes in functional characteristics can be further improved and/or manipulated when implemented in selected IgG isotypes. Similarly, the native characteristics of an isotype Fc can be manipulated by one or more amino acid modifications. Many IgG isotypes (i.e., IgG1, IgG2, IgG3, and IgG4) have different serum half-life, complement binding, FcγR binding affinity, and effector function activity (e.g. ADCC) due to differences in the amino acid sequence of their hinge and/or Fc regions. , CDC), and exhibit different physical and functional characteristics.

In one embodiment, amino acid modifications and IgG Fc regions are independently selected based on their respective binding and/or effector function activities to engineer a bispecific antibody with desired characteristics. In certain embodiments, amino acid modifications and IgG hinge/Fc regions are separately assayed for binding and/or effector function activity as described herein or known in the art for IgG1. In one embodiment, the amino acid modification and the IgG hinge/Fc region provide a similar function in a bispecific antibody or other Fc-containing molecule (e.g., immunoglobulin), such as reduced ADCC activity (or other effector function) and /or shows increased binding to FcγRIIB. In another embodiment, the invention includes variant Fc regions and selected IgG regions comprising combinations of amino acid modifications known in the art, which exhibit new properties, wherein the modifications and/or regions are described herein. It is not detected when analyzed independently as shown.

One way to determine the binding affinity of an antibody to CD3 is by measuring the binding affinity of a monofunctional Fab fragment of the antibody. To obtain monofunctional Fab fragments, antibodies (e.g., IgG) can be cleaved with papain or expressed recombinantly. The affinity of the CD3 Fab fragment of the antibody was determined using pre-immobilized streptavidin using HBS-EP running buffer (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v surfactant P20). Surface plasmon resonance (BIACORE™ 3000™ surface plasmon resonance (SPR) system with sensor chip (SA) or anti-mouse Fc or anti-human Fc, BIACORE™, INC., Piscataway, NJ, USA. It can be determined by the material)). Biotinylated or Fc-fused human CD3 is diluted into HBS-EP buffer to a concentration of less than 0.5 μg/mL and injected across individual chip channels using variable contact times to produce two ranges of antigen densities, i.e., detailed kinetics. 50 to 200 response units (RU) can be achieved for research or 800 to 1,000 RU for screening assays. Regeneration studies showed that 25 mM NaOH in 25% v/v ethanol effectively removed bound Fab while maintaining the activity of CD3 on the chip for over 200 injections. Typically, serial dilutions of purified Fab samples (sequential concentrations from 0.1 to 10x assumed K _D ) are injected at 100 μL/min for 1 min, and a dissociation time of up to 2 h is allowed. The concentration of Fab protein is determined by ELISA and/or SDS-PAGE electrophoresis using an unknown concentration of Fab (determined by amino acid analysis) as a reference. Dynamic association rates (k _on ) and dissociation rates (k _off ) were calculated using the BIAevaluation program using the 1:1 Langmuir association model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B. (1994). Methods Enzymology 6. 99-110]) are simultaneously obtained by comprehensively fitting the data. The equilibrium dissociation constant (K _D ) value is calculated as k _off /k _on . This protocol measures the binding affinity of antibodies to any CD3, including human CD3, CD3 of another mammal (e.g., mouse CD3, rat CD3, or primate CD3), and various forms of CD3 (e.g., glycosylated CD3). Suitable for use in determining degrees. The binding affinity of an antibody is generally measured at 25°C, but can also be measured at 37°C.

Antibodies described herein can be produced by any method known in the art. For the production of hybridoma cell lines, the route and scheme of immunization of the host animal is generally maintained by established and conventional techniques for antibody stimulation and production, as further described herein. General techniques for the production of human and mouse antibodies are known to those skilled in the art and/or described herein.

It is contemplated that any mammalian organism, including humans, or antibody producing cells therefrom, may be engineered to serve as a basis for the production of mammalian, including human, and hybridoma cell lines. Typically, the host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, intrapodially and/or intradermally with a dose of immunogen, including those described herein.

Hybridomas can be produced from lymphocytes and immortalized myeloma cells using the general somatic cell hybridization technique of Kohler, B. and Milstein, C., Nature 256:495-497, 1975, or as described in Buck, D. W., et al., In Vitro, 18:377-381, 1982]. Available myeloma cell lines, such as, but not limited to, X63-Ag8.653 and cell lines from the Salk Institute, Cell Distribution Center (San Diego, CA) can be used for hybridization. Generally, the technique involves fusing myeloma cells and lymphoid cells using a fusogen such as polyethylene glycol, or by electrical means well known to those skilled in the art. After fusion, the cells are separated from the fusion medium and grown in a selective growth medium such as hypoxanthine-aminopterin-thymidine (HAT) medium to remove unhybridized parental cells. Any of the media described herein, with or without serum supplementation, can be used to culture hybridomas secreting any monoclonal antibody. Another alternative to cell fusion techniques (EBV immortalized B cells) can be used to produce the monoclonal antibodies of the invention. Hybridomas were expanded and subcloned as needed, and supernatants were assayed for anti-immunogenic activity by routine immunoassay procedures (e.g., radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay).

Hybridomas that can be used as a source of antibodies encompass all derivative, progeny cells of the parent hybridoma that produce monoclonal antibodies specific for CD3, or portions thereof.

Hybridomas producing such antibodies can be grown in vitro or in vivo using known procedures. Monoclonal antibodies can be isolated from culture media or body fluids, as required, by conventional immunoglobulin purification procedures, such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration. If present, unwanted activity can be removed, for example, by performing precipitation on an adsorbent prepared with the immunogen attached to a solid phase and eluting or releasing the desired antibody from the immunogen. Bifunctional or derivatizing agents, such as maleimidobenzoyl sulfosuccinimide ester (conjugated via a cysteine residue), N-hydroxysuccinimide (via a lysine residue), glutaraldehyde, succinic anhydride, SOCl ₂ or Using R ¹ N=C=NR, where R and R ¹ are different alkyl groups, a protein that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin or soy trypsin inhibitor A host animal is immunized with a fragment of human CD3, containing the target amino acid sequence conjugated to.

If desired, the antibody of interest can be sequenced and the polynucleotide sequence can then be cloned into a vector for expression or propagation. The sequence encoding the antibody of interest can be maintained in a vector in host cells, which can then be expanded and frozen for future use. Production of recombinant monoclonal antibodies in cell culture can be accomplished through cloning of antibody genes from B cells by means known in the art (e.g., Tiller et al., J. Immunol. Methods, 329, 112 , 2008]; see US 7,314,622).

In the alternative, polynucleotide sequences can be used for genetic engineering to humanize the antibody or improve the affinity or other characteristics of the antibody. For example, the constant region can be engineered to more resemble the human discomfort region to avoid an immune response if the antibody is used in human clinical trials and treatments. It may be desirable to genetically engineer the antibody sequence to obtain greater affinity for CD3 and greater therapeutic potency.

In some embodiments, the antibody has a constant region modified to eliminate or reduce Fc gamma receptor binding. For example, the Fc may be human IgG2 containing mutation D265, where the amino acid residues are numbered relative to the wild-type IgG2 sequence. Accordingly, in some embodiments, the constant region has a modified constant region having the sequence shown in SEQ ID NO:80. In some embodiments, the antibody has a modified constant region having the sequence shown in SEQ ID NO:81.

There are four general steps to humanize a monoclonal antibody. (1) determination of the nucleotides and predicted amino acid sequences of the starting antibody light and heavy chain variable regions, (2) design of the humanized antibody, i.e. determination of the antibody framework regions to be used during the humanization process, (3) actual humanization methodology/technique, and (4) transfection and expression of humanized antibodies. For example, US 4,816,567; 5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761; 5,693,762; 5,585,089; and 6,180,370.

Many humanized antibody molecules with antigen binding sites derived from non-human immunoglobulins have been described, including chimeric antibodies with rodent or modified rodent V regions and their associated CDRs fused to human constant regions. For example, Winter et al. Nature 349:293-299, 1991], [Lobuglio et al. Proc. Nat. Acad. Sci. USA 86:4220-4224, 1989], [Shaw et al. J Immunol. 138:4534-4538, 1987] and [Brown et al. Cancer Res. 47:3577-3583, 1987]. Other references describe rodent CDRs grafted into a human supporting framework region (FR) prior to fusion with the appropriate human antibody constant region. For example, Riechmann et al. Nature 332:323-327, 1988], [Verhoeyen et al. Science 239:1534-1536, 1988] and [Jones et al. Nature 321:522-525, 1986. Another reference describes rodent CDRs supported by recombinantly engineered rodent framework regions. See, for example, EP 0519596. These “humanized” molecules are designed to minimize unwanted immunological responses to rodent anti-human antibody molecules, which would limit the duration and effectiveness of therapeutic application of these moieties in human recipients. For example, an antibody constant region can be engineered to be immunologically inactive (e.g., not trigger complement lysis). For example, PCT/GB99/01441; See UK 9809951.8. Other methods of humanization of antibodies that can be used are described in Daugherty et al., Nucl. Acids Res. 19:2471-2476, 1991], US 6,180,377; 6,054,297; 5,997,867; 5,866,692; 6,210,671; and 6,350,861; and WO 01/27160.

Additionally, the principles relating to humanized antibodies discussed above can be used, for example, in other mammals (e.g. humans, non-human primates, murine, donkey, sheep, rabbit, goat, guinea pig, camel, horse or chicken). It is applied to custom antibodies for: Additionally, one or more aspects of humanization of the antibodies described herein, such as CDR grafting, framework mutation, and CDR mutation, can be combined.

In one variation, fully human antibodies can be obtained by using commercially available mice engineered to express specific human immunoglobulin proteins. Additionally, transgenic animals designed to produce more desirable (e.g., fully human antibodies) or stronger immune responses can be used for the production of humanized or human antibodies. Examples of such technologies include TED (Medarex, Inc., Princeton, NJ, USA).

In the alternative, the antibody can be produced recombinantly and expressed using any method known in the art. In another alternative, antibodies can be produced recombinantly by phage display technology. For example, US 5,565,332; 5,580,717; 5,733,743; and 6,265,150; and Winter et al., Annu. Rev. Immunol. 12:433-455, 1994]. Alternatively, in vitro human antibodies and antibody fragments from immunoglobulin variable (V) domain gene repertoires from unimmunized donors using phage display technology (McCafferty et al., Nature 348:552-553, 1990). can produce. According to this technique, antibody V domain genes are cloned in frame into the major or minor coat protein genes of filamentous bacteriophages, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because filamentous particles contain a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also leads to selection of genes encoding antibodies that exhibit these properties. Thus, phages mimic some of the characteristics of B cells. Phage display can be performed in a variety of formats; For a review see, for example, Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571, 1993. Several sources of V-gene segments can be used for phage display. Clackson et al., Nature 352:624-628, 1991 isolated a diverse array of anti-oxazolone antibodies from a small, randomly assembled library of V genes derived from the spleens of immunized mice. A repertoire of V genes from unimmunized human donors can be constructed, and antibodies to a diverse array of antigens (including auto-antigens) can be prepared as described in Mark et al., J. Mol. Biol. 222:581-597, 1991] or [Griffith et al., EMBO J. 12:725-734, 1993]. In a natural immune response, antibody genes rapidly accumulate mutations (somatic hypermutation). Some of the changes introduced will confer higher affinity, and B cells expressing high affinity surface immunoglobulins will preferentially replicate and differentiate during subsequent antigen challenge. This natural process can be mimicked using a technique known as “chain shuffling” (Marks et al., Bio/Technol. 10:779-783, 1992). In this method, the affinity of a “primary” human antibody obtained by phage display is increased by sequentially replacing the heavy and light chain V domain genes with a repertoire of naturally occurring variants of V domain genes obtained from unimmunized donors. It can be improved. This technology allows the production of antibodies and antibody fragments with affinities ranging from pM to nM. Strategies for producing very large phage antibody repertoires (also known as “largest libraries”) are described in Waterhouse et al., Nucl. Acids Res. 21:2265-2266, 1993. Gene shuffling can also be used to derive human antibodies from rodent antibodies, where the human antibodies have similar affinity and specificity as the starting rodent antibody. According to this method, also referred to as "epitope imprinting", the heavy or light chain V domain genes of rodent antibodies obtained by phage display technology are replaced with a repertoire of human V domain genes to produce rodent-human chimeras. Selection against an antigen results in the isolation of a human variable region that can restore a functional antigen binding site, i.e. the epitope dictates (imprints) partner selection. When this process is repeated to replace the remaining rodent V domain, a human antibody is obtained (see WO 93/06213). Unlike conventional humanization of rodent antibodies by CDR grafting, this technique provides fully human antibodies that do not have framework or CDR residues of rodent origin.

Antibodies can be produced recombinantly by first isolating the antibody and antibody-producing cells from the host animal to obtain the genetic sequence, and then using the genetic sequence to recombinantly express the antibody in the host cell (e.g., CHO cell). . Another method that can be used is to express the antibody sequences in plants (e.g. tobacco) or transgenic milk. A method for recombinantly expressing antibodies in plants or milk is disclosed. See, for example, Peeters, et al. Vaccine 19:2756, 2001]; [Lonberg, N. and D. Huszar Int. Rev. Immunol 13:65, 1995]; and Pollock, et al., J Immunol Methods 231:147, 1999. Methods for producing derivatives of antibodies, e.g., humanized, single chain, etc., are known in the art.

Additionally, immunoassays and flow cytometry sorting techniques, such as fluorescence activated cell sorting (FACS), can be used to isolate antibodies specific for CD3 or the corresponding antigen.

The antibodies described herein can be bound to many different solid supports or carriers. These supports may be active and/or inactive. Known supports include polypropylene, polystyrene, polyethylene, dextran, nylon, amylase, glass, native and modified cellulose, polyacrylamide, agarose and magnetite. The nature of the support may be soluble or insoluble for the purposes of the present invention. Those skilled in the art will know of other suitable supports for binding antibodies, or can determine these using routine experimentation. In some embodiments, the scaffold comprises a moiety that targets the myocardium.

DNA encoding monoclonal antibodies is readily isolated and sequenced using routine procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells serve as a preferred source of such DNA. After isolation, the DNA can be placed into an expression vector (such as that disclosed in WO 87/04462) and then transferred to a host cell that does not otherwise produce immunoglobulin proteins, such as Escherichia coli cells, simian COS cells, CHO cells. Alternatively, composites of monoclonal antibodies can be obtained in recombinant host cells by transfection into myeloma cells. See, for example, WO 87/04462. Additionally, DNA can be modified, for example, by substituting the coding sequences for human heavy and light chain constant regions for the homologous murine sequences (Morrison et al., Proc. Nat. Acad. Sci. 81:6851, 1984) or -The coding sequence for an immunoglobulin polypeptide may be modified by covalently linking all or part of the immunoglobulin coding sequence. In this way, “chimeric” or “hybrid” antibodies are produced that have the binding specificity of the monoclonal antibodies herein.

CD3 or other antigen antibodies described herein are identified or characterized by methods known in the art in which a decrease in the level of CD3 or other antigen expression is detected and/or measured. In some embodiments, CD3 antibodies are identified by incubating a candidate agent with CD3 and monitoring binding and/or concomitant decrease in the level of CD3 expression. Binding assays are performed using purified CD3 polypeptides or cells that naturally or transfected express CD3 polypeptides. In one embodiment, the binding assay is a competitive binding assay, in which the ability of a candidate antibody to compete with a known CD3 antibody for CD3 binding is assessed. The assay can be performed in a variety of formats, including ELISA format.

After initial confirmation, the activity of the candidate CD3 or other antigen antibody can be further confirmed and improved by known biological assays to test the desired biological activity. Alternatively, biological assays can be used to screen candidates directly. Some of the methods for identifying and characterizing antibodies are described in detail in the Examples.

CD3 or other antigen antibodies can be characterized using methods well known in the art. For example, one way is to identify the epitope it binds to, or epitope mapping. Analysis of crystal structures of antibody-antigen complexes, competition, as described, for example, in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999. Many methods are known in the art for mapping and characterizing the location of epitopes on proteins, including assays, gene fragment expression assays, and synthetic peptide-based assays. In a further example, epitope mapping can be used to determine the sequence to which an antibody binds. Epitope mapping is commercially available from various sources, such as PepScan Systems (Edelhertweg 15, 8219 PH Lelystad, The Netherlands). Epitopes may be linear epitopes (i.e. contained in a single stretch of amino acids), or conformational epitopes (formed by three-dimensional interactions of amino acids that may not necessarily be contained in a single stretch). Peptides of various lengths (e.g., at least 4 to 6 amino acids in length) can be isolated or synthesized (e.g., recombinantly) and used in binding assays using CD3 or antigen antibodies. In another example, the epitope to which a CD3 or other antigen antibody binds can be determined in a systematic screening by using overlapping peptides derived from the CD3 or antigen sequence and determining binding by the CD3 or other antigen antibody. Following gene fragment expression analysis, the open reading frame encoding CD3 or another antigen is fragmented randomly or by specific gene constructs and determines the reactivity of the antibody to be tested with the expressed fragment of CD3 or other antigen. Gene fragments can be produced, for example, by PCR, and then transcribed and translated into proteins in the presence of radioactive amino acids in vitro. Binding of the antibody to radiolabeled CD3 or other antigen fragments is then determined by immunoprecipitation and gel electrophoresis. Additionally, specific epitopes can be identified by using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries). Alternatively, defined libraries of overlapping peptide fragments can be tested for binding to the test antibody in a simple binding assay. In additional examples, mutagenesis of the antigen binding domain, domain exchange experiments and alanine scanning mutagenesis can be performed to identify residues that are required and/or sufficient and/or necessary for epitope binding. For example, domain exchange experiments allow various fragments of CD3 or other antigenic proteins to match the sequence of an antigen from another species (e.g., mouse), or to a closely related but antigenically distinct protein (e.g., Trop This can be done by using mutant CD3 or other antigens replaced by -1). By assessing the binding of antibodies to mutant CD3 or other antigens, the importance of specific CD3 or other antigen fragments for antibody binding can be assessed.

Another method that can be used to characterize a CD3 or other antigen antibody is to determine whether the CD3 or other antigen antibody each binds to the same epitope as another antibody known to bind the same antigen, i.e. It is a competition assay using various fragments on CD3 or other antigens. Competitive analysis is well known to those skilled in the art.

Expression vectors can be used for direct expression of CD3 or other antigen antibodies. Those skilled in the art are familiar with the administration of expression vectors to obtain expression of exogenous proteins in vivo. For example, US 6,436,908; 6,413,942; and 6,376,471. Administration of expression vectors includes topical or systemic administration (including injection), oral administration, particle gun or catheterized administration, and topical. In another embodiment, the expression vector is administered directly to the sympathetic trunk or ganglion or into the coronary artery, atrium, ventricle, or pericardium.

Additionally, targeted delivery of therapeutic compositions containing expression vectors or subgenomic polynucleotides can be used. Receptor-mediated DNA transfer techniques are described, for example, in Findeis et al., Trends Biotechnol., 11:202, 1993; [Chiou et al., Gene Therapeutics: Methods And Applications Of Direct Gene Transfer, J.A. Wolff, ed., 1994]; [Wu et al., J. Biol. Chem., 263:621, 1988]; [Wu et al., J. Biol. Chem., 269:542, 1994]; [Zenke et al., Proc. Natl. Acad. Sci. USA, 87:3655, 1990]; and [Wu et al., J. Biol. Chem., 266:338, 1991. Therapeutic compositions containing polynucleotides are administered in amounts ranging from about 100 ng to about 200 mg of DNA for topical administration in gene therapy protocols. Additionally, concentration ranges of DNA from about 500 ng to about 50 mg, from about 1 μg to about 2 mg, from about 5 μg to about 500 μg, and from about 20 μg to about 100 μg DNA can be used during gene therapy protocols. Therapeutic polynucleotides and polypeptides can be delivered using gene delivery vehicles. Gene delivery vehicles may be of viral or non-viral origin (see generally Jolly, Cancer Gene Therapy, 1:51, 1994; Kimura, Human Gene Therapy, 5:845, 1994; Connelly, Human Gene Therapy, 1:185, 1995]; and [Kaplitt, Nature Genetics, 6:148, 1994]). Expression of these coding sequences can be driven using endogenous mammalian or heterologous promoters. Expression of the coding sequence may be constitutive or regulated.

Virus-based vectors for delivery of the polynucleotide of interest and expression in the desired cell are well known in the art. Exemplary virus-based vehicles include, but are not limited to, recombinant retroviruses (e.g., WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91 /02805; US 5,219,740 and 4,777,127; GB 2,200,651; EP 0 345 242), alphavirus-based vectors (e.g., Sindbis virus vector, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Los Liber virus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), and adeno-associated virus (AAV) vectors ( See, for example, WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655). Curiel, Hum. Administration of DNA linked to killed adenovirus can also be used, as described in Gene Ther., 3:147, 1992.

Also, polycationic condensed DNA linked or unlinked to a single dead adenovirus (see, e.g., Curiel, Hum. Gene Ther., 3:147, 1992); Ligand-linked DNA (see, e.g., Wu, J. Biol. Chem., 264:16985, 1989); Eukaryotic delivery vehicles non-viral, including nuclear charge neutralization or fusion with cells (see, e.g., US 5,814,482; WO 95/07994; WO 96/17072; WO 95/30763; and WO 97/42338) and cell membranes Delivery vehicles and methods may be used. Additionally, naked DNA can be used. Exemplary naked DNA introduction methods are described in WO 90/11092 and US 5,580,859. Liposomes that can act as gene delivery vehicles are described in US 5,422,120; WO 95/13796; WO 94/23697; WO 91/14445; and EP 0524968. Additional approaches are described in Philip, Mol. Cell Biol., 14:2411, 1994] and [Woffendin, Proc. Natl. Acad. Sci., 91:1581, 1994.

In some embodiments, a composition comprising a pharmaceutical composition comprising an antibody of the invention as described herein or produced by a method and having the characteristics described herein. As used herein, the pharmaceutical composition may comprise one or more antibodies that bind to a tumor antigen, one or more bispecific antibodies that bind to a CD3 tumor antigen, and/or one or more polynucleotides comprising sequences encoding one or more of these antibodies. You can. These compositions may further comprise suitable excipients, including buffering agents well known in the art, such as pharmaceutically acceptable excipients.

In one embodiment, the invention features a composition that is a combination of a CD3 antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with a CD3 antibody. Exemplary agents that can be advantageously combined with CD3 antibodies include, but are not limited to, other agents that bind to CD3 and/or activate CD3 signaling (including other antibodies or antigen-binding fragments thereof, etc.) and/or bind directly to CD3. Includes agents that activate or stimulate immune cell activation, but do not The invention further encompasses additional combination therapies and co-formulations involving the CD3 antibodies of the invention. Additionally, the present invention provides methods for producing any of these antibodies. Bispecific antibodies of the invention can be produced by procedures known in the art, including de novo protein synthesis and recombinant expression of nucleic acids encoding binding proteins. The nucleic acid sequence of interest can be produced by recombinant methods (e.g., PCR mutagenesis of previously produced variants of the polynucleotide of interest) or by solid-phase DNA synthesis. Typically, recombinant expression methods are used. In one aspect, the invention provides polynucleotides comprising sequences encoding CD3 VH and/or VL. Due to the degeneracy of the genetic code, a variety of nucleic acid sequences encode each immunoglobulin amino acid sequence, and the present invention includes all nucleic acids encoding the binding proteins described herein.

Additionally, they can be produced in vitro using known synthetic protein chemistry methods, including the involvement of chimeric or hybrid antibody cross-linkers. For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Reagents suitable for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

In recombinant humanized antibodies, the Fcγ portion can be modified to avoid interaction of Fcγ receptors with complement and the immune system. The technology for producing such antibodies is described in WO 99/58572. For example, the constant region can be engineered to more closely resemble a human constant region to avoid an immune response when the antibody is used in human clinical trials and treatments. See, for example, US 5,997,867 and 5,866,692.

The CD3 antibodies disclosed herein may contain one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequence from which the antibody is derived. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The invention includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, wherein at least one amino acid in one or more framework and/or CDR regions is a corresponding residue of the germline sequence from which the antibody is derived, and Mutated to a corresponding residue in another human germline sequence, or by a conservative amino acid substitution of a corresponding germline residue (such sequence changes are collectively referred to herein as “germline mutations”). One skilled in the art can readily produce many antibodies and antigen-binding fragments containing one or more distinct germline mutations or combinations thereof starting from the heavy and light chain variable region sequences disclosed herein. In certain embodiments, all framework and/or CDR residues within the VH and/or VL domains are back mutated to residues found in the original germline sequence from which the antibody was derived. In some embodiments, only certain residues are mutated, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2, or CDR3. Mutations occur back to the original germline sequence. In some additional embodiments, one or more of the framework and/or CDR residues are mutated to a corresponding residue in a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived).

Additionally, the antibodies of the invention may contain any combination of two or more germline mutations within the framework and/or CDR regions, for example, wherein certain distinct residues are mutated to corresponding residues in a particular germline sequence. On the other hand, certain other residues that differ from the original germline sequence are retained or mutated to corresponding residues in the different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations may exhibit one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or functional biological properties (if (according to this), can be tested for reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained by these general methods are included within the present invention.

The invention also includes CD3 antibodies comprising variants of any of the HC VR, LC VR and/or CDR amino acid sequences disclosed herein with one or more conservative substitutions. For example, the present invention provides conservative amino acids, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc., compared to any HC VR, LC VR, and/or CDR amino acid sequence disclosed herein. Includes CD3 antibodies having HC VR, LC VR, and/or CDR amino acid sequences with substitutions.

Accordingly, the invention encompasses modifications to antibodies and polypeptides of the invention, including functionally equivalent antibodies that do not significantly affect their properties, and variants with enhanced or decreased activity and/or affinity. . For example, the amino acid sequence can be mutated to obtain an antibody with the desired binding affinity for tumor antigen and/or CD3. Modification of polypeptides is routine practice in the art and need not be described in detail herein. Examples of modified polypeptides include conservative substitutions of amino acid residues, deletion of one or more amino acids, or addition of amino acids that do not significantly deleteriously change the functional activity or mature (enhance) the affinity of the polypeptide for the ligand, or use of chemical analogs. It includes polypeptides having.

Amino acid sequence insertions include amino- and/or carboxy-terminal fusions ranging in length from one residue to polypeptides containing more than a hundred residues, and intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue or antibodies fused to an epitope tag. Other insertion variants of the antibody molecule include fusions to the N- or C-terminus of the antibody with enzymes or polypeptides that increase the half-life of the antibody in the blood circulation.

Substitution variants have one or more amino acid residues removed from the antibody molecule and a different residue inserted in that position. Sites of greatest interest for substitution mutagenesis include hypervariable regions, but FR changes are also considered. Conservative substitutions are shown in Table 6 under the heading “Conservative Substitutions.” If such substitutions result in changes in biological activity, more substantial changes, indicated as “exemplary substitutions” in Table 6 or described further below with respect to amino acid classes, can be introduced and the product screened.

[Table 6]

Substantial modification of the biological properties of the antibody may result from maintaining (a) the structure of the polypeptide backbone at the site of the substitution, such as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chains. This is achieved by selecting substitutions that differ significantly in their effect on . Naturally occurring amino acid residues are divided into groups based on common side chain properties:

(1) Nonpolar: Norleucine, Met, Ala, Val, Leu, Ile;

(2) Polar without charge: Cys, Ser, Thr, Asn, Gln;

(3) Acidic (negatively charged): Asp, Glu;

(4) Basic (positively charged): Lys, Arg;

(5) Residues affecting chain orientation: Gly, Pro;

(6) Aromatic: Trp, Tyr, Phe, His.

Non-conservative substitutions are performed by exchanging members of one of these classes for another class.

Additionally, any cysteine residues that are not involved in maintaining the proper conformation of the antibody can be replaced, usually with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine linkages can be added to an antibody to improve its stability, especially if the antibody is an antibody fragment, such as an Fv fragment.

Amino acid modifications can range from changing or modifying one or more amino acids to complete redesign of a region, such as a variable region. Changes in the variable region can alter binding affinity and/or specificity. In some embodiments, no more than 1 to 5 conservative amino acid substitutions are made within the CDR domain. In some embodiments, no more than 1 to 3 conservative amino acid substitutions are made within the CDR domain. In another embodiment, the CDR domain is VH CDR3 and/or VL CDR3.

Modifications also include glycosylated and non-glycosylated polypeptides, polypeptides with other post-translational modifications (such as glycosylation with various sugars, acetylation, and phosphorylation). Antibodies are glycosylated at conserved positions in their constant regions (Jefferis and Lund, Chem. Immunol. 65:111-128, 1997; Wright and Morrison, TibTECH 15:26-32, 1997). The oligosaccharide side chains of immunoglobulins are involved in protein functions (Boyd et al., Mol. Immunol. 32:1311-1318, 1996; Wittwe and Howard, Biochem. 29:4175-4180, 1990) and sugars. Intermolecular interactions between parts of a protein, which can affect the shape and three-dimensional surface on which the glycoprotein is presented (Jefferis and Lund, supra; Wyss and Wagner, Current Opin. Biotech. 7:409 -416, 1996]). In addition, oligosaccharide can act to target the presented glycoprotein to a specific molecule based on a specific recognition structure. Additionally, glycosylation of antibodies has been reported to affect antibody-dependent cellular cytotoxicity (ADCC). In particular, CHO cells with tetracycline-controlled expression of β(1,4)-N-acetylglucosaminyltransferase III (GnTIII) (glycosyltransferase-catalyzed formation of bipartite GlcNAc) improved ADCC activity. It has been reported to do so (Umana et al., Mature Biotech. 17:176-180, 1999]).

Glycosylation of antibodies is typically N-linked or O-linked. “N-linked” refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine, asparagine-X-threonine and asparagine-X-cysteine, where Therefore, the presence of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation involves one of the sugars N-acetylgalactosamine, galactose or xylose on a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used. refers to the attachment of

The addition of glycosylation sites to an antibody is conveniently accomplished by altering the amino acid sequence, so that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). Additionally, alterations may be effected by addition or substitution by one or more serine or threonine residues to the sequence of the original antibody (in the case of O-linked glycosylation sites).

Additionally, the glycosylation pattern of an antibody can be altered without altering the underlying nucleotide sequence. Glycosylation is highly dependent on the host cell used to express the antibody. Since the cell types used for expression of recombinant glycoproteins, e.g. antibodies, as potential therapeutics are rarely native cells, modifications of the glycosylation pattern of the antibody may be expected (see, e.g. Hse et al., J. Biol. Chem. 272:9062-9070, 1997].

In addition to the choice of host cells, factors that affect glycosylation during recombinant production of antibodies include growth mode, media formulation, culture density, oxygenation, pH, purification scheme, etc. Various methods, including the introduction or overexpression of specific enzymes involved in oligosaccharide production, have been proposed to alter the glycosylation pattern achieved in a particular host organism (US 5,047,335; 5,510,261 and 5,278,299). Glycosylation, or certain types of glycosylation, uses, for example, endoglycosidase H (Endo H), N-glycosidase F, endoglycosidase F1, endoglycosidase F2, endoglycosidase F3. Thus, it can be removed from the glycoprotein by enzymes. Additionally, recombinant host cells can be genetically engineered to be defective in processing certain types of polysaccharides. These and similar techniques are well known in the art.

Other methods of modification include the use of coupling techniques known in the art, including but not limited to enzymatic means, oxidative substitution, and chelation. Modifications may be used, for example, for attachment of labels for immunoassays. Modified polypeptides can be produced using procedures established in the art and screened using standard assays known in the art, some of which are described below and in the Examples.

In some embodiments of the invention, the antibody has a modified constant region, such as increased affinity for the human Fc gamma receptor; Immunologically inactive or partially inactive, for example, does not trigger complement-mediated lysis, does not stimulate antibody-dependent cell-mediated cytotoxicity (ADCC), or does not activate macrophages; a constant region with reduced activity (compared to an unmodified antibody) in any one or more of triggering complement-mediated lysis, stimulating antibody-dependent cell-mediated cytotoxicity (ADCC), or activating microglial cells. . Different modifications of the constant regions can be used to achieve optimal levels and/or combinations of effector functions. See, for example, Morgan et al., Immunology 86:319-324, 1995; [Lund et al., J. Immunology 157:4963-9 157:4963-4969, 1996]; [Idusogie et al., J. Immunology 164:4178-4184, 2000]; [Tao et al., J. Immunology 143: 2595-2601, 1989]; and [Jefferis et al., Immunological Reviews 163:59-76, 1998]. In some embodiments, the constant region is described in Eur. J. Immunol., 29:2613-2624, 1999]; PCT/GB99/01441; and/or as modified as described in UK 9809951.8. In another embodiment, the constant region is non-glycosylated relative to N-linked glycosylation. In some embodiments, the constant region is non-glycosylated relative to N-linked glycosylation by mutating the glycosylated amino acid residues or flanking residues that are part of the N-glycosylation recognition sequence. For example, N-glycosylation site N297 can be mutated to A, Q, K or H. Tao et al., J. Immunology 143: 2595-2601, 1989; and [Jefferis et al., Immunological Reviews 163:59-76, 1998]. In some embodiments, the constant region is non-glycosylated relative to N-linked glycosylation. The constant region is non-glycosylated relative to N-linked glycosylation, either enzymatically (e.g., carbohydrate removal by the enzyme PNGase) or by expression in glycosylation-deficient host cells.

Other antibody modifications may include antibodies modified as described in WO 99/58572. These antibodies comprise, in addition to a binding domain directed to the target molecule, an effector domain having an amino acid sequence substantially homologous to all or part of the constant region of a human immunoglobulin heavy chain. These antibodies can bind target molecules without triggering significant complement-dependent lysis or cell-mediated destruction of the target. In some embodiments, the effector domain is capable of specifically binding FcRn and/or FcγRIIb. It is typically based on a chimeric domain derived from two or more human immunoglobulin heavy chain CH2 domains. Antibodies modified in this way are particularly suitable for use in long-term antibody therapy to avoid inflammation and other side effects of conventional antibody therapy.

The present invention includes affinity matured embodiments. For example, affinity matured antibodies can be produced by procedures known in the art (Marks et al., Bio/Technology, 10:779-783, 1992; Barbas et al., Proc. Nat. Acad. Sci, USA 91:3809-3813, 1994]; [Schier et al., Gene, 169:147-155, 1995]; [Yelton et al., J. Immunol., 155:1994-2004, 1995]; [Jackson et al., J. Immunol., 154(7):3310-9, 1995]; [Hawkins et al., J. Mol. Biol., 226:889-896, 1992]; and WO 2004/058184).

The following methods can be used to adjust the affinity of antibodies and characterize CDRs. One method of characterizing the CDRs of an antibody and/or altering (e.g., improving) the binding affinity of a polypeptide, such as an antibody, is referred to as “library scanning mutagenesis.” Generally, library scanning mutagenesis works as follows. One or more amino acid positions in the CDR can be selected from two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17) using methods known in the art. , 18, 19 or 20) amino acids are replaced. This creates a small library of clones (in some embodiments, one at every amino acid position analyzed), each with a complexity of two or more members (where two or more amino acids are substituted at all positions). Typically, the library also contains clones containing natural (unsubstituted) amino acids. A small number of clones from each library, e.g., about 20 to 80 clones (depending on the complexity of the library), are screened for binding affinity to the target polypeptide (or other binding target) and are subjected to increased, equal, or decreased binding affinity. Candidates with or without the bond are identified. Methods for determining binding affinity are well known in the art. Binding affinity can be determined using BIACORE™ surface plasmon resonance analysis, which detects differences in binding affinity of about 2-fold or greater. BIACORE™ is particularly useful when the starting antibody binds with a relatively high affinity, e.g., with a K _D of about 10 nM or less. Screening using BIACORE™ surface plasmon resonance is described in the Examples herein.

Binding affinity can be determined using Kinexa Biocensor, scintillation proximity assay, ELISA, ORIGEN immunoassay (IGEN), fluorescence quenching, fluorescence transfer, and/or yeast display. Additionally, binding affinity can be screened using a suitable biological assay.

In some embodiments, all amino acid positions in a CDR are replaced (in some embodiments, one by one) by all 20 natural amino acids using mutagenesis methods known in the art (some of which are described herein). This creates a small library of clones (in some embodiments, one for every amino acid position analyzed) with a complexity of 20 members each (if all 20 amino acids are substituted at all positions).

In some embodiments, the library to be screened contains substitutions at two or more positions that may be present in the same CDR or in two or more CDRs. Accordingly, a library may contain substitutions at more than one position in one CDR. A library may contain substitutions at two or more positions in two or more CDRs. The library may contain substitutions in 3, 4, or 5 or more positions, which positions are found in 2, 3, 4, 5, or 6 CDRs. Substitutions can be made using low-redundancy codons. See, for example, Table 2 of Balint et al., Gene 137(1):109-18, 1993.

Candidates with improved binding can be sequenced to identify CDR substitution mutants that result in improved affinity (also referred to as “improved” substitutions). Additionally, candidates that bind can be sequenced to identify CDR substitutions that maintain binding.

Multiple rounds of screening may be performed. For example, a candidate with improved binding (each comprising an amino acid substitution at one or more positions in one or more CDRs) may also have at least at each improved CDR position (i.e., an amino acid position in the CDR for which the substitution mutant exhibits improved binding). It is useful for the design of a second library containing the original and substituted amino acids. Production, and screening or selection of such libraries are discussed further below.

Additionally, library scanning mutagenesis is useful insofar as the frequency of clones with improved binding, identical binding, reduced binding, or no binding provides information regarding the importance of each amino acid position for the stability of the antibody-antigen complex. , provides a means for characterizing CDRs. For example, if a position in a CDR maintains binding when changed to all 20 amino acids, then that position is identified as a position unlikely to be required for antigen binding. Conversely, if a position in a CDR maintains binding with only a small percentage of substitutions, that position is identified as being important for CDR function. Accordingly, library scanning mutagenesis methods generate information about the positions of CDRs that can vary by many different amino acids (including all 20 amino acids), and the positions of CDRs that are unchanged or can vary by only a few amino acids.

Candidates with improved affinity can be combined with the original amino acids at that position in a second library containing the improved amino acid and, depending on the complexity of the library desired or acceptable, using the desired screening or selection method. Additional substitutions may additionally be included. Additionally, if necessary, adjacent amino acid positions may be randomized into at least two or more amino acids. Randomization of adjacent amino acids allows for additional conformational flexibility in mutant CDRs, which in turn may allow or enable the introduction of large numbers of improving mutations. Additionally, the library may contain substitutions at positions that did not result in improved affinity in the first round of screening.

The second library comprises screening using BIACORE™ surface plasmon resonance analysis, and selection using any method known in the art for selection, including phage display, yeast display, and ribosome display. Using any method known in the art, library members are screened and selected for having improved and/or altered binding affinity.

Antibody isolation and antibody production techniques are as follows. However, it is understood that antibodies can be produced from or incorporated into other polypeptides using techniques known in the art. For example, nucleic acids encoding a polypeptide of interest (e.g., a ligand, receptor, or enzyme) can be isolated from a cDNA library produced from a tissue thought to have polypeptide mRNA and express it at detectable levels. The library is screened using probes (e.g., antibodies or oligonucleotides of about 20 to 80 bases) designed to identify the gene or protein encoded by the gene of interest. Screening of cDNA or genomic libraries using selected probes is performed using standard procedures described in Chapters 10 to 12 of Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). It can be done.

CD3 antibodies for the treatment of autoimmune disorders

In one aspect, a method of treating an autoimmune disorder in an individual is provided, comprising administering to an individual in need thereof an effective amount of a composition comprising an antibody described herein.

Autoimmune disorders as used herein include, but are not limited to, inflammatory bowel disease, systemic lupus erythematosus, rheumatoid arthritis, diabetes (type I), multiple sclerosis, Addison's disease, celiac disease, dermatomyositis, Graves' disease, Hashimoto's thyroiditis, Hashimoto's encephalopathy. , myasthenia gravis, reactive arthritis, Sjogren's syndrome, atopic allergy, atopic dermatitis, autoimmune enteropathy, autoimmune hepatitis, autoimmune lymphoproliferative syndrome, autoimmune peripheral neuropathy, autoimmune pancreatitis, autoimmune polyendocrine syndrome, autoimmune progesterone. Dermatitis, autoimmune urticaria, autoimmune uveitis, Behcet's disease, Castleman's disease, cold agglutination disease, Crohn's disease, dermatomyositis, eosinophilic fasciitis, gastrointestinal pemphigoid, Goodpasture syndrome, Guillian-Barre syndrome, hidradenitis suppurativa, lethargy, Includes pemphigus vulgaris, polymyositis, relapsing polychondritis, rheumatic fever, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease, and vasculitis Wegener's granulomatosis.

Multispecific antibodies and uses thereof

CD3 antibodies of the invention may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. See, for example, Tutt et al., 1991, J. Immunol. 147:60-69]; [Kufer et al., 2004, Trends Biotechnol. 22:238-244]. CD3 antibodies of the invention may be linked to or co-expressed with another functional molecule, for example another peptide or protein. For example, an antibody or fragment thereof is functionally linked (e.g., by chemical coupling, gene fusion, non-covalent association, etc.) to one or more other molecular entities, such as another antibody or antibody fragment, to have a second binding specificity. Multispecific antibodies can be produced.

In one aspect, the antibodies of the invention are multispecific antibodies. In certain embodiments, the CD3 antibody is a bispecific antibody that specifically binds human CD3. In some such embodiments, the bispecific antibody further binds specifically to a tumor antigen. In another such embodiment, the bispecific antibodies of the invention simultaneously target T cells (CD3) and tumor cells and successfully induce and activate T cell cytotoxicity against tumor cells expressing tumor antigens.

In additional embodiments of each of the foregoing, the bispecific antibody that binds CD3 is characterized by any one or more of the following characteristics: (a) binding a specific tumor antigen in an individual to a disease associated with malignant cells (e.g. treating, preventing or ameliorating one or more symptoms of a B cell-related cancer (e.g., multiple myeloma); (b) inhibiting tumor growth or progression in an individual (having a malignant tumor expressing a specific tumor antigen); (c) inhibiting metastasis of cancer (malignant) cells expressing a specific tumor antigen in an individual (having one or more malignant cells expressing a specific tumor antigen); (d) inducing regression (e.g., organ regression) of tumors expressing tumor antigens; (e) exerting cytotoxic activity on malignant cells expressing tumor antigens; (f) increasing progression-free survival in individuals with tumor-related disorders; (g) increasing overall survival of individuals with tumor-related disorders; (h) reducing the use of additional chemotherapeutic or cytotoxic agents in individuals with tumor-related disorders; (i) reducing tumor burden in individuals with tumor-related disorders; or (j) blocking the interaction of unidentified factors with tumor antigens.

As used herein, a bispecific antibody of the invention refers to a complex of two or more polypeptide chains each comprising at least one antibody VL region and one antibody VH region or fragments thereof, wherein each polypeptide The VL and VH regions of the chain originate from different antibodies. In specific aspects, bispecific antibodies comprise dimers or tetramers of polypeptide chains containing both VL and VH regions. Individual polypeptide chains comprising a multimeric protein may be covalently linked to one or more other peptides of the multimer by interchain disulfide bonds.

In some such embodiments, the bispecific antibodies of the invention comprise a first heterodimer-promoting domain on a first polypeptide chain and a second heterodimer-promoting domain on a second polypeptide chain (Figure 1). Taken together, the first and second heterodimer-promoting domains induce heterodimerization (e.g., by interaction of knobs and holes on complementary heterodimer-promoting domains) and/or generate bispecific antibodies. It acts to stabilize and/or stabilize the bispecific antibody. In some embodiments, the first heterodimer-promoting domain and the second heterodimer-promoting domain each comprise a CH2 domain and a CH3 domain, wherein the amino acid sequence of each CH2 domain and/or each CH3 domain is heterologous. Modified to induce merization and/or stabilize the bispecific antibody.

In some embodiments, the first heterodimer-promoting domain may comprise an Fc chain with CH2 and/or CH3 domains modified to include knobs (protrusions) or holes (cavities). In some such embodiments, the amino acid sequence of the CH2 domain and/or CH3 domain comprises one or more amino acid modifications such that (a) the CH3 domain of the first heterodimer-promoting domain forms a knob; (b) The CH3 domain of the second heterodimer-promoting domain forms a hole. In another such embodiment, the CH3 domain of the first heterodimer-promoting domain comprises the mutations Y349C and/or T366W to form a knob; The CH3 domain of the second heterodimer-promoting domain contains mutations S354C, T366S, L368A and/or Y407V to form a hole (numbering according to EU index).

In one embodiment, when the second heterodimer-promoting domain comprises a CH2 and/or CH3 domain modified to include a hole, the first heterodimer-promoting domain comprises the sequence of SEQ ID NO: 78 It may include CH2 and/or CH3 domains modified to include knobs. In another embodiment, when the second heterodimer-promoting domain comprises CH2 and/or CH3 domains modified to include knobs, the first heterodimer-promoting domain has the sequence of SEQ ID NO:79. Includes a hall (cavity) that contains.

Each polypeptide chain of a bispecific antibody comprises a VL region and a VH region that can be covalently linked by a glycine-serine linker (linker 1 or linker 2) containing glycine and serine residues, thereby rendering the antibody binding domain self- Restricted from assembly. Additionally, each polypeptide chain includes a heterodimerization domain that promotes heterodimerization and/or stabilization of multiple polypeptide chains and reduces the likelihood of homodimerization of different polypeptide chains. The heterodimerization domain may be located at the N-terminus or C-terminus of the polypeptide chain. The heterodimerization domain may comprise a cysteine linker (linker 3) having a length of 1, 2, 3, 4, 5, or 6 or more amino acid residues. The interaction of two polypeptide chains can create two VL/VH pairs, creating two epitope binding domains, i.e. a bivalent molecule. The VH or VL regions are not limited to any position within the polypeptide chain (i.e. not limited to the amino terminus or the carboxy terminus), nor are the regions limited to their relative positions relative to each other, i.e. the VL region has It can be the N-terminus, and vice versa. The only limitation is that complementary polypeptide chains are available for production of functional bispecific antibodies. If the VL and VH regions are derived from antibodies specific for different antigens, production of a functional bispecific antibody requires the interaction of two different polypeptide chains (i.e. production of a heterodimer). In contrast, when two different polypeptide chains do not interact (e.g. in a recombinant expression system), i.e. one contains VLA and VHB (A is the first epitope, B is the second epitope) and the other When one contains VLB and VHA, two different binding sites are created: VLA-VHA and VLB-VHB. In all bispecific antibody polypeptide chain pairs, misalignment or misalignment of the two chains (e.g., interaction of VL-VL or VH-VH regions) is possible. However, purification of functional bispecific antibodies is readily performed based on the immunospecificity of the appropriately dimerized binding site using any affinity known in the art or exemplified herein, for example, affinity chromatography. do.

In one embodiment, the polypeptide chain of a bispecific antibody may include various linkers and peptides. Linkers and peptides can be 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9 or more amino acids.

In some embodiments, domain 1 is covalently linked to a first heterodimer-promoting domain via a cysteine linker and domain 2 is covalently linked to a second heterodimer-promoting domain via a cysteine linker. Each cysteine linker contains one or more cysteine residues to allow for intramolecular disulfide bonding. In a further embodiment of each of the preceding, the cysteine linker (linker 3) comprises at least 5 amino acids.

In some embodiments, the first polypeptide chain is covalently linked to the second polypeptide chain by one or more disulfide bonds. In some such embodiments, one or more disulfide bonds are formed between Linker 3 of the first polypeptide chain and Linker 3 of the second polypeptide chain. In another such embodiment, one or more disulfide bonds are formed between the first heterodimer-promoting domain and the second heterodimer-promoting domain. In specific embodiments, each disulfide bond is formed by joining two cysteine residues. In one aspect of the invention, the bispecific antibody comprises a first polypeptide chain and a second polypeptide chain, as shown in Figure 1. In some such embodiments, linker 3 may comprise a truncated human IgG1 lower hinge region having the sequence of SEQ ID NO:64 and one or more glycine residues preceding the lower hinge region.

Bispecific antibodies of the invention are capable of binding two separate and distinct epitopes simultaneously. In certain embodiments, the one or more epitope binding sites are specific for the CD3 determinant expressed on immune effector cells, such as expressed on T lymphocytes. In one embodiment, the bispecific antibody molecule binds to effector cell determinants and also activates the effector cell.

In one aspect, the invention provides a first polypeptide chain and a second polypeptide chain. In one embodiment, the first polypeptide chain comprises the amino acid sequence set forth as SEQ ID NO:36. In another embodiment, the second polypeptide chain comprises the amino acid sequence set forth as one or more of SEQ ID NO: 37 or 38 (Table 7). In a preferred embodiment, the first polypeptide chain comprises the amino acid sequence set forth as SEQ ID NO:36; The second polypeptide chain comprises the amino acid sequence set forth as SEQ ID NO: 37 or 38. In one embodiment, the second polypeptide chain is any polypeptide that can associate with the first polypeptide chain through contact points. Table 7 shows the sequences of the first and second polypeptide chains for the GUCY2c-H2B4 and GUCY2c-2B5 bispecific antibodies.

[Table 7]

In certain embodiments, the bispecific antibodies of the invention (a) bind to the extracellular domain of a human tumor antigen; (b) exhibits prolonged serum and tumor half-life of 30 minutes to 100 days; and/or (c) an EC ₅₀ value of 0.0001 to 100 nM in the presence of increased levels of tumor expression or increased levels of receptor density.

In one embodiment, the epitope binding domain is suitable for treating breast cancer, ovarian cancer, thyroid cancer, prostate cancer, uterine cancer, lung cancer (including, but not limited to, non-small cell lung cancer and small cell lung cancer), bladder cancer, endometrial cancer, head and neck cancer, testicular cancer, and glial cancer. It can bind to tumor-related antigens associated with subspecies and digestive cancer. Cancers of the digestive system include, but are not limited to, esophageal cancer, stomach cancer, small intestine cancer, colon cancer, rectal cancer, anal cancer, liver cancer, gallbladder cancer, appendix cancer, bile duct cancer, and pancreatic cancer. In specific embodiments, the treatment activates a cytolytic T cell response.

Effector deletion mutants of human IgG1 CH2-CH3

The Fc chain of human IgG1 was modified using standard primer-guided PCR mutagenesis to introduce mutations L234A, L235A and G237A (SEQ ID NO: 82, numbering according to EU index), thereby deleting the effector function due to binding to FcγRIII. Effector function deletion phenotypes are provided (Canfield et al., J. Exp. Med (1991) 173: 1483-1491; Shields et al., J. Biol. Chem. (2001) 276:6591-604 ]).

Knob-in-hole mutations of human IgG1 CH2-CH3

Knob-in-hole is an effective design strategy known in the art for engineering antibody heavy chain homodimers for heterodimerization. In this approach, 'knob' variants are obtained by replacing small amino acids with larger ones in one chain of the Fc chain of IgG1 (e.g. Y349C and T366W) (numbering according to EU index). 'Knobs' are designed to be inserted into 'holes' in the CH3 domain of the complementary chain of the Fc chain (e.g. S354C, T366S, L368A and Y407V) by replacing large residues with smaller ones (numbering according to EU index).

In some embodiments, as provided in Table 8, complementary mutations are introduced to induce heterodimerization of the resulting Fc chains, such that each Fc chain has one set of mutations, i.e., the knobs (or protrusions) of the Fc chain. ) in Y349C and T366W (SEQ ID NO: 78), or in the hole (or cavity) of the Fc chain at S354C, T366S, L368A and Y407V (SEQ ID NO: 79). When co-transfected into a suitable mammalian host, the DNA encoding the amino acid sequences of SEQ ID NOs: 78 and 79 is bispecific, with one knob (or protrusion) Fc chain associated with one hole (or hollow) Fc chain. Produces an Fc domain, an antibody.

[Table 8]

The CD3-tumor antigen bispecific antibody of the invention is stable against aggregation in thermal stability studies and a strong bispecific antibody-Fc fusion targeting both human CD3 and tumor antigen. The knob-in-hole Fc domain allows for improved expression and improved purification in CHO cells, resulting in high purity of the desired heterodimer. Mutations engineered within the Fc domain abolish FcγR binding, potentially avoiding ADCC-mediated T cell exhaustion. Additionally, incorporation of the Fc domain into a bispecific antibody enhances the stability of the molecule as shown by differential scanning calorimetry (DSC).

Bispecific antibodies can be engineered to contain one or more cysteine residues that can interact with corresponding cysteine residues on another polypeptide chain of the invention to form interchain disulfide bonds. Interchain disulfide bonds can act to stabilize bispecific antibodies, improving expression and recovery in recombinant systems, resulting in stable and consistent formulations, as well as improving the stability of the isolated and/or purified product in vivo. Cysteine residues can be introduced into any part of the polypeptide chain, either as a single amino acid or as part of a larger amino acid sequence, such as a hinge region. In a specific aspect, one or more cysteine residues are engineered to occur at the C-terminus of the polypeptide chain.

The present invention includes methods and compositions for the treatment, prevention or management of cancer in an individual, comprising administering to the individual a therapeutically effective amount of an antibody engineered according to the invention, wherein the molecule is added to a cancer antigen. Combine with Antibodies of the invention may be particularly useful for preventing, inhibiting or reducing the growth and/or regression of primary tumors and metastasis of cancer cells. Without being limited to a specific mechanism of action, the antibodies of the invention may mediate effector functions, which may result in tumor clearance, tumor reduction, or a combination thereof.

In one aspect, the present invention provides a therapeutic treatment method for stimulating T cell activation using a CD3 antibody of the antibody of the present invention, wherein the treatment method uses a therapeutically effective amount of a pharmaceutical composition comprising the antibody of the present invention. It involves administering to an individual in need. The disorder to be treated is any disease or condition that is ameliorated, improved, inhibited or prevented by stimulation of CD3 activity or signaling. In specific embodiments, the invention provides bispecific antigen binding molecules, e.g., bispecific antibodies that bind CD3 and a target antigen.

In one aspect, the invention provides a method of inhibiting tumor growth, comprising contacting the tumor with an effective amount of an antibody that binds CD3 (including each antibody described herein).

In another aspect, the invention provides a method of inhibiting tumor growth in an individual, comprising administering to the individual an effective amount of an antibody that binds CD3 (including each antibody described herein).

In another aspect, the invention provides a method of modulating angiogenesis in an individual, comprising administering to the individual an effective amount of an antibody that binds to CD3 (including each antibody described herein).

In another aspect, the invention provides a method of reducing tumor formation in an individual, comprising administering to the individual an effective amount of an antibody that binds CD3 (including each antibody described herein).

In one aspect, the invention provides a method of treating a disease associated with tumor antigen expression in an individual. Thus, tumor antigens engineer bispecific antibodies to immunospecifically recognize tumor antigens and CD3 antigens on T cells. Bispecific antibodies engineered according to the present invention are useful for the prevention or treatment of cancer because they have cytotoxic activity by induced CD3 antibody-induced activated killer T cells.

In certain embodiments, the present invention provides methods of treating cancer. In specific embodiments, the cancer is breast cancer, ovarian cancer, thyroid cancer, prostate cancer, uterine cancer, lung cancer (including, but not limited to, non-small cell lung cancer and small cell lung cancer), bladder cancer, endometrial cancer, head and neck cancer, testicular cancer, glioblastoma, or It is a cancer of the digestive system. In certain embodiments, the cancer of the digestive system is selected from the group consisting of esophageal cancer, stomach cancer, small intestine cancer, colon cancer, rectal cancer, anal cancer, liver cancer, gallbladder cancer, appendix cancer, bile duct cancer, and pancreatic cancer.

In one aspect, the invention provides an antibody, bispecific antibody, or pharmaceutical composition disclosed herein for use in therapy. In certain embodiments, the invention also provides CD3 bispecific antibodies for use in a method of treating cancer as defined herein. In specific embodiments, the treatment activates a cytolytic T cell response.

Also provided is an antibody or bispecific antibody disclosed herein in the manufacture of a medicament for use in the treatment of the invention. In some embodiments, the treatment is treatment of cancer. In some embodiments, the cancer is breast cancer, ovarian cancer, thyroid cancer, prostate cancer, uterine cancer, lung cancer (including, but not limited to, non-small cell lung cancer and small cell lung cancer), bladder cancer, endometrial cancer, head and neck cancer, testicular cancer, glioblastoma, and It is selected from the group consisting of cancers of the digestive system. In certain embodiments, the cancer of the digestive system is selected from the group consisting of esophageal cancer, stomach cancer, small intestine cancer, colon cancer, rectal cancer, anal cancer, liver cancer, gallbladder cancer, appendix cancer, bile duct cancer, and pancreatic cancer.

In one aspect, the invention provides a polynucleotide encoding an antibody or bispecific antibody disclosed herein. In another embodiment, the invention provides vectors comprising the polynucleotides disclosed herein. In another embodiment, the invention provides host cells comprising the vectors disclosed herein. In some such embodiments, the host cell recombinantly produces an antibody or bispecific antibody disclosed herein. In specific embodiments, the host cell is selected from the group consisting of bacterial cell lines, mammalian cell lines, insect cell lines, and yeast cell lines, and in vitro cell-free protein synthesis systems. In certain embodiments, the mammalian cell line is a CHO cell line.

In one aspect, a method of treating cancer in an individual in need thereof is provided, comprising a) providing a bispecific antibody described herein, and b) administering the bispecific antibody to the individual.

In some embodiments, a method of inhibiting tumor growth or progression in an individual having malignant cells expressing a tumor antigen is provided, comprising administering to said individual in need thereof an effective amount of a composition comprising an antibody described herein. It includes administration to an individual. In some embodiments, a method of inhibiting metastatic cells expressing a tumor antigen in an individual is provided, comprising administering to the individual in need thereof an effective amount of a composition comprising an antibody described herein. In some embodiments, a method of inducing tumor regression of malignant cells in an individual is provided, comprising administering to an individual in need thereof an effective amount of a composition comprising an antibody described herein.

In specific aspects, an antibody of the invention inhibits the growth of cancer cells by at least 99%, at least 95%, at least 90%, or at least 85% compared to the growth of cancer cells in the absence of the antibody or bispecific antibody of the invention. , at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20% or at least Suppress or reduce by 10%.

In specific aspects, the antibody is at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, compared to the absence of the antibody or bispecific antibody of the invention. Kill cells or inhibit the growth of cancer cells by at least 45%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. Suppress or reduce.

In one aspect, the invention provides a bispecific antibody described herein for the treatment of a disease (e.g., cancer) associated with tumor antigen expression in an individual in need thereof. Provides an effective amount of the composition (e.g., pharmaceutical composition) comprising it.

In another aspect, the invention provides an antibody described herein for use in the treatment of a disease (e.g., cancer) associated with tumor antigen expression in an individual in need thereof. to provide. In some embodiments, provided is a superbody described herein for inhibition of tumor growth or progression in an individual having malignant cells expressing tumor antigens. In some embodiments, an antibody described herein is provided for inhibition of metastasis of malignant cells expressing a tumor antigen in an individual in need thereof. In some embodiments, provided are antibodies described herein for inducing tumor regression in an individual with malignant cells expressing a tumor antigen.

In another aspect, the invention provides the use of an antibody described herein in the manufacture of a medicament for the treatment of a disease associated with tumor antigen expression (e.g., cancer). In some embodiments, use of an antibody described herein is provided in the manufacture of a medicament for inhibiting tumor growth or progression. In some embodiments, provided is the use of an antibody described herein in the manufacture of a medicament for inhibiting metastasis of malignant cells expressing tumor antigens. In some embodiments, use of an antibody described herein is provided in the manufacture of a medicament for inducing tumor regression.

In some embodiments, the methods described herein further include treating the individual with another form of therapy. In some embodiments, the additional form of therapy is additional anti-cancer therapy, including but not limited to chemotherapy, radiation, surgery, hormone therapy, and/or additional immunotherapy.

The present invention relates to administering the molecules of the invention in combination with other therapies known to those skilled in the art for the treatment or prevention of cancer, including, but not limited to, current standard experimental chemotherapy, biological therapy, immunotherapy, radiotherapy or surgery. It additionally includes: In some aspects, the molecules of the invention may be administered in combination with a therapeutically or prophylactically effective amount of one or more agents, therapeutic antibodies, or other agents known to those skilled in the art for the treatment and/or prevention of cancer.

Accordingly, a method of treating cancer includes administering an effective amount of a multispecific antibody (e.g., a bispecific antibody) of the invention in combination with a chemotherapeutic agent to an individual in need thereof. These combination treatments may be administered separately, sequentially, or simultaneously.

The dosages and frequencies of administration provided herein are encompassed by the terms “therapeutically effective and prophylactically effective.” Dosage and frequency may vary depending on factors specific to each individual, including the specific therapeutic or prophylactic agent administered, the severity and type of cancer, the route of administration, and the individual's age, weight, response, and past medical history. A suitable regimen can be selected by the person skilled in the art by taking into account the above factors and according to the dosages reported and recommended, for example, in Physician's Desk Reference (56th ed., 2002).

Therefore, in addition to redirecting T cells to tumor-specific antigens, bispecific T cell binding molecules are also used to deliver other diagnostic or therapeutic compounds to tumor-expressing cells on the surface. Accordingly, the bispecific T cell binding molecule will be attached directly or indirectly, for example via a linker, to the drug, which will be delivered directly to the tumor-bearing cells. Therapeutics include, but are not limited to, compounds such as nucleic acids, proteins, peptides, amino acids or derivatives, glycoproteins, radioisotopes, lipids, carbohydrates, or recombinant viruses. Nucleic acid therapeutic and diagnostic moieties are shared with, but not limited to, antisense nucleic acids, single or duplex DNA. Oligonucleotides derived for cross-linking, and triplex forming oligonucleotides.

pharmaceutical composition

The present invention also provides compositions comprising a therapeutically effective amount of an antibody disclosed herein and a pharmaceutically acceptable carrier.

The antibodies of the present invention may be in the form of a pharmaceutical composition for administration formulated to suit the selected mode of administration and pharmaceutically acceptable diluents or excipients such as buffers, surfactants, preservatives, solubilizers, isotonic agents, stabilizers, carriers, etc. You can. Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton Pa., 18th ed., 1995 provides a compendium of formulation techniques as are generally known to those skilled in the art.

These pharmaceutical compositions can be administered by any method known in the art that generally achieves its intended purpose of treating cancer. The route of administration may be parenteral, as defined herein, which refers to modes of administration including, but not limited to, intravenous, intramuscular, intraperitoneal, subcutaneous, and intra-articular injection and infusion. Methods of administering and dosing molecules (e.g., antibodies, pharmaceutical compositions) according to the present invention include the type of disease to be treated or, appropriately, its stage, the antigen to be controlled, the type of coexisting treatment, the frequency of treatment, if present, and the desired effect. nature, and depends on the individual's weight, age, health, diet and sex. Accordingly, the dosage regimens used in practice may vary greatly and thus may deviate from the dosage regimens presented herein.

Various formulations of the antibodies of the invention may be used for administration. In some embodiments, the antibody may be administered neat. In some embodiments, the antibody and pharmaceutically acceptable excipients may exist in various formulations. Pharmaceutically acceptable excipients are known in the art and are relatively inert substances that enable administration of pharmacologically effective substances. For example, an excipient can provide form or consistency or act as a diluent. Suitable excipients include, but are not limited to, stabilizers, wetting and emulsifying agents, salts for adjusting osmotic pressure, encapsulating agents, buffering agents, and skin penetration enhancers. Excipients and formulations for parenteral and non-enteral drug delivery are described in Remington, The Science and Practice of Pharmacy 21st Ed. Mack Publishing, 2005].

In some embodiments, these agents are formulated for administration by injection (e.g., intraperitoneally, intravenously, subcutaneously, intramuscularly, etc.). Accordingly, these agents may be combined with pharmaceutically acceptable vehicles such as saline, Ringer's solution, dextrose solution, etc. The specific dosage regimen, i.e., dosage, timing and repetition, will depend on the particular individual and that individual's medical history.

Antibodies described herein can be administered using any suitable method, including injection (e.g., intraperitoneally, intravenously, subcutaneously, intramuscularly, etc.). Additionally, antibodies, such as monoclonal antibodies or bispecific antibodies, can be administered via inhalation as described herein. Generally, for administration of an antibody of the invention, the dosage depends on the host being treated and the particular mode of administration. In one embodiment, the dosage range for an antibody of the invention will be from about 0.001 to about 20,000 μg/kg of body weight. The term “body weight” is applicable when the patient is receiving treatment. When treating isolated cells, “body weight” as used herein refers to “total cell weight.” The term “total body weight” may be used and applies to both isolated cells and patient treatment. All concentrations and treatment levels are expressed herein as “body weight” or simply “kg” and are also considered to include “whole cell body weight” and “total body weight”. However, those skilled in the art will recognize various dosage ranges, for example, 0.01 to 20,000 μg/kg body weight, 0.02 to 15,000 μg/kg body weight, 0.03 to 10,000 μg/kg body weight, 0.04 to 5,000 μg/kg body weight, 0.05 to 2,500 μg/kg. Body weight, 0.06 to 1,000 μg/kg body weight, 0.07 to 500 μg/kg body weight, 0.08 to 400 μg/kg body weight, 0.09 to 200 μg/kg body weight, or 0.1 to 100 μg/kg body weight. Additionally, those skilled in the art will know, for example, 0.0001 μg/kg, 0.0002 μg/kg, 0.0003 μg/kg, 0.0004 μg/kg, 0.005 μg/kg, 0.0007 μg/kg, 0.001 μg/kg, 0.1 μg/kg, 1.0 μg/kg. kg, 1.5 μg/kg, 2.0 μg/kg, 5.0 μg/kg, 10.0 μg/kg, 15.0 μg/kg, 30.0 μg/kg, 50 μg/kg, 75 μg/kg, 80 μg/kg, 90 μg/ kg, 100 μg/kg, 120 μg/kg, 140 μg/kg, 150 μg/kg, 160 μg/kg, 180 μg/kg, 200 μg/kg, 225 μg/kg, 250 μg/kg, 275 μg/kg kg, 300 μg/kg, 325 μg/kg, 350 μg/kg, 375 μg/kg, 400 μg/kg, 450 μg/kg, 500 μg/kg, 550 μg/kg, 600 μg/kg, 700 μg/ kg, 750 μg/kg, 800 μg/kg, 900 μg/kg, 1 μg/kg, 5 μg/kg, 10 μg/kg, 12 μg/kg, 15 mg/kg, 20 mg/kg and/or 30 μg/kg It will be appreciated that a variety of different dosage levels of mg/kg will be used. All of these dosages are exemplary and any dosage between these points is also expected to be used in the present invention. Any of the above dosage ranges or dosage levels may be used for the antibodies of the invention. For repeated administration of several days or more, depending on the condition, treatment is continued until the desired suppression of symptoms occurs or a sufficient therapeutic level is achieved, e.g. to inhibit or delay tumor growth/progression or metastasis of cancer cells. It lasts.

Additionally, other dosage regimens may be useful depending on the pattern of pharmacokinetic decay that one of ordinary skill in the art wishes to achieve. In one embodiment, the antibody of the invention is administered at an initial starting dose, followed by higher doses and/or continuous, substantially constant doses. In some embodiments, 1 to 4 administrations per week are contemplated. In some embodiments, administration once per month, once every two months, or once every three months is contemplated. The progress of such therapy is readily monitored by routine techniques and analyses. Dosage regimens may vary over time.

For the purposes of the present invention, an appropriate dosage of an antibody will depend on the antibody or composition thereof used, the type and severity of the condition to be treated, whether the agent is administered for therapeutic purposes, previous therapy, the patient's history and response to the agent, and the type of agent administered. It will depend on the patient's clearance of the agent and the discretion of the attending physician. Typically, clinicians will administer the antibody until the dosage achieves the desired outcome. Dosage and/or frequency may vary during the course of treatment. Empirical considerations, such as half-life, will generally contribute to the determination of dosage. For example, antibodies that are compatible with the human immune system, such as humanized antibodies or fully human antibodies, can be used to extend the half-life of an antibody and prevent the antibody from being attacked by the host's immune system. The frequency of administration may be determined and adjusted during the course of treatment and is generally, but need not be, based on the treatment and/or suppression and/or improvement and/or delay of symptoms, for example, tumor growth inhibition or delay, etc. does not exist. Alternatively, sustained, sustained release formulations of the antibody may be appropriate. A variety of formulations and devices for achieving sustained release are known in the art.

In one embodiment, the dosage of the antibody can be determined empirically in an individual receiving one or more doses of the antibody. The subject receives incremental doses of antibody. To evaluate effectiveness, indicators of disease can be followed.

In certain embodiments, administration of the antibody inhibits tumor growth, tumor regression, reduces tumor size, reduces tumor cell number, delays tumor growth, abscopal effect, inhibits tumor metastasis, reduces metastatic lesions over time, and acts as a chemotherapeutic agent. or causes one or more effects selected from the group consisting of reduced use of cytotoxic agents, reduced tumor burden, increased progression-free survival, increased overall survival, complete response, partial and stable disease.

Administration of antibodies according to the methods of the invention may be continuous or intermittent, depending, for example, on the physiological state of the recipient, whether the purpose of administration is therapeutic or prophylactic, and other factors known to those skilled in the art. Administration of the antibody may be essentially continuous over a preselected period of time or may be carried out as a series of spaced doses.

In some embodiments, more than one antibody may be present. There may be at least 1, at least 2, at least 3, at least 4, at least 5 or more different antibodies. In general, they may have complementary activities that do not adversely affect each other. For example, one or more of the following antibodies may be used: a first CD3 antibody directed to one epitope on CD3, and a second CD3 antibody directed to a different epitope on CD3.

In some embodiments, the antibodies herein are particularly useful for parenteral administration, such as intravenous administration or administration into a body cavity or organ lumen.

Therapeutic formulations of antibodies used in accordance with the present invention may contain proteins of the desired purity in the presence of optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington, The Science and Practice of Pharmacy 21st Ed. Mack Publishing, 2005). It can be prepared in the form of a lyophilized formulation or aqueous solution for storage by mixing with. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, and organic acids; salts such as sodium chloride; Antioxidants such as ascorbic acid, and methionine; Preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclo hexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; Proteins such as serum albumin, gelatin or immunoglobulins; Hydrophilic polymers such as polyvinylpyrrolidone; Amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates such as glucose, mannose, or dextrins; Chelating agents such as EDTA; Sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (eg Zn-protein complexes); and/or nonionic surfactants such as TWEEN™, Pluronics™ or polyethylene glycol (PEG).

Liposomes containing antibodies can be prepared by methods known in the art (e.g., Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang, et al., Proc. Natl Acad. Sci. USA 77:4030 (1980)] and US 4,485,045 and 4,544,545). Liposomes with enhanced circulation time are disclosed in US 5,013,556. Particularly useful liposomes can be produced by reverse phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through a filter of defined pore size to produce liposomes with the desired diameter.

Additionally, the active ingredient may be microcapsules prepared, for example, by coacervation technology or by interfacial polymerization, for example, hydroxymethylcellulose, or gelatin-microcapsules and poly-(methylmethacrylate). ) can be entrapped in microcapsules, colloidal drug delivery systems (e.g. liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or microemulsions. These techniques are described in Remington, The Science and Practice of Pharmacy 21st Ed. Mack Publishing, 2005].

Sustained-release preparations can be prepared. Suitable examples of sustained release formulations include a semipermeable matrix of solid hydrophobic polymer containing the antibody, the matrix being in the form of a shaped article, for example a film or microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (e.g. poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactide (US 3,773,919), L-glutamic acid, and 7 ethyl. -Copolymers of L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT (trade mark) (injectable consisting of lactic acid-glycolic acid copolymer and leuprolide acetate) microspheres), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

Formulations used for in vivo administration must be sterile. This is easily achieved, for example, by filtration through sterile filtration membranes. The therapeutic antibody (eg, CD3 antibody) composition is typically placed in a container with a sterile access port, such as an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle.

The compositions according to the invention may be in unit dosage form, such as tablets, pills, capsules, powders, granules, solutions or suspensions or suppositories, for oral, parenteral or rectal administration, or for administration by inhalation or inhalation.

For the preparation of solid compositions, such as tablets, the main active ingredient is combined with a pharmaceutical carrier, for example customary tabletting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gum. , and other pharmaceutical diluents, such as water, to form a solid pre-formulated composition containing a homogeneous mixture of a compound of the present invention or a non-toxic pharmaceutically acceptable salt thereof. When these pre-formulated compositions are referred to as homogeneous, this means that the active ingredients are evenly distributed throughout the composition so that the composition can be readily subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules. The solid pre-formulated composition is then subdivided into unit dosage forms of the type described above containing from 0.1 to about 500 mg of the active ingredient of the invention. Tablets or pills of the compositions of the present invention may be coated or mixed to provide dosage forms that provide the advantage of delayed action. For example, a tablet or pill may contain an inner dose and an outer dose component, the latter in the form of an envelope over the former. The two components may be separated by an enteric layer, which serves to resist degradation in the stomach and allow the internal components to be delivered intact to the duodenum or to delay their release. A variety of materials can be used in these enteric layers or coatings, including many polymeric acids and mixtures of polymeric acids with such materials such as shellac, cetyl alcohol, and cellulose acetate.

Suitable surface active agents are in particular non-ionic agents such as polyoxyethylene sorbitan (e.g. Tween® 20, 40, 60, 80 or 85) and other sorbitans (e.g. Span® 20, 40). , 60, 80 or 85). Compositions with surface active agents may conveniently include 0.05 to 5% surface active agent, which may be 0.1 to 2.5%. It will be understood that other ingredients, such as mannitol or other pharmaceutically acceptable vehicles, may be added as needed.

Compositions within the scope of the present invention have the antibody present in an amount effective to achieve the desired medical effect for the treatment of cancer. Individual needs may vary from patient to patient, but determination of the optimal range of effective amounts of all ingredients is within the ability of a clinician of ordinary skill.

Additionally, examples of such compositions, and methods of formulation, are described in the sections above and below. In some embodiments, the composition includes one or more antibodies. In some embodiments, the antibody is a human antibody, humanized antibody, or chimeric antibody. In some embodiments, the antibody comprises a constant region capable of triggering the desired immune response, such as antibody-mediated lysis or ADCC. In some embodiments, the antibody comprises a constant region that does not trigger an unwanted or undesirable immune response, such as antibody-mediated lysis or ADCC.

It is understood that the composition may comprise more than one antibody, such as CD3 or a mixture of CD3 and CD3 antibodies that recognize different epitopes of tumor antigens. Other exemplary compositions include more than one antibody that recognizes the same epitope of antibodies from different species or binds different epitopes of CD3 (eg, human CD3).

In some embodiments, the antibody may be administered in combination with the administration of one or more additional therapeutic agents. This includes, but is not limited to, biotherapeutics and/or chemotherapeutics (such as, but not limited to, vaccines), CAR-T cell-based therapy, radiation therapy, cytokine therapy, CD3 bispecific antibodies, inhibitors of other immunosuppressive pathways, and angiogenesis agents. Inhibitors, T cell activators, inhibitors of metabolic pathways, mTOR inhibitors, inhibitors of the adenosine pathway, tyrosine kinase inhibitors (including but not limited to Inlyta), ALK inhibitors and sunitinib, BRAF inhibitors, epigenetic modifications agents, IDO1 inhibitors, JAK inhibitors, STAT inhibitors, cyclin-dependent kinase inhibitors, biotherapeutics (including but not limited to VEGF, VEGFR, EGFR, Her2/neu, other growth factor receptors, CD40, CD-40L, CTLA-4, OX-40 , antibodies to 4-1BB, TIGIT and ICOS), immunogenic agents (e.g. attenuated cancerous cells, tumor antigens, antigen-presenting cells such as dendritic cells pulsed with tumor-derived antigens or nucleic acids, immunostimulatory cytokines ( For example, IL-2, IFNα2, GM-CSF), and cells transfected with genes encoding immunostimulatory cytokines (such as, but not limited to, GM-CSF).

Examples of biotherapeutics include therapeutic antibodies, immunomodulators, and therapeutic immune cells.

Therapeutic antibodies can have specificity for a variety of antigens. For example, a therapeutic antibody may be directed to a tumor-associated antigen, such that binding of the antibody to the antigen promotes death of cells expressing the antigen. In another example, the therapeutic antibody may be directed to an antigen (e.g. PD-1) on immune cells, preventing down-regulation of the activity of cells expressing the antigen (thereby promoting the activity of cells expressing the antigen) ). In some cases, therapeutic antibodies may act through a number of different mechanisms (for example, they may i) promote the death of cells expressing the antigen, and ii) activate immune cells that the antigen has contacted with the cells expressing the antigen. can prevent it from causing downregulation).

Therapeutic antibodies may be directed to, for example, antigens listed below. For some antigens, exemplary antibodies directed to the antigen are also included below (in parentheses after the antigen). Additionally, the following antigens may be referred to herein as “target antigens” and the like. Target antigens for therapeutic antibodies herein include, for example: 4-1BB (eg, utomilumab); 5T4; A33; alpha-folate receptor 1 (e.g. mirvetuximab soravtansine); Alk-1; BCMA [e.g. PF-06863135 (see US 9,969,809)]; BTN1A1 (see for example WO 2018/222689); CA-125 (e.g. abagovomab); Carboanhydrase IX; CCR2; CCR4 (e.g. mogamulizumab); CCR5 (e.g. leronlimab); CCR8; CD3 [e.g. blinatumomab (CD3/CD19 bispecific), PF-06671008 (CD3/P-cadherin bispecific), PF-06863135 (CD3/BCMA bispecific), PF- 07062119 (CD3/GUCY2c bispecific)]; CD19 (e.g. blinatumomab, MOR208); CD20 (e.g. ibritumomab tiuxetan, obinutuzumab, ofatumumab, rituximab, ublituximab); CD22 (inotuzumab ozogamicin, moxetumomab pasudotox); CD25; CD28; CD30 (e.g. brentuximab vedotin); CD33 (e.g. gemtuzumab ozogamicin); CD38 (e.g. daratumumab, isatuximab), CD40; CD-40L; CD44v6; CD47; CD52 (e.g. alemtuzumab); CD63; CD79 (e.g. polatuzumab vedotin); CD80; CD123; CD276/B7-H3 (e.g. omburtamab); CDH17; CEA; ClhCG; CTLA-4 (e.g. ipilimumab, tremelimumab), CXCR4; Desmoglein 4; DLL3 (e.g. rovalpituzumab tesirine); DLL4; E-cadherin; EDA; EDB; EFNA4; EGFR (e.g. cetuximab, depatuxizumab mafodotin, necitumumab, panitumumab); EGFRvIII; endosialin; EpCAM (e.g. oportuzumab monatox); FAP; fetal acetylcholine receptor; FLT3 (see for example WO 2018/220584); GD2 (e.g. dinutuximab, 3F8); GD3; GITR; GloboH; GM1; GM2; GUCY2C (e.g. PF-07062119); HER2/neu [e.g. margetuximab, pertuzumab, trastuzumab; ado-trastuzumab emtansine, trastuzumab duocarmazine, PF-06804103 (see US 8,828,401)]; HER3; HER4; ICOS; IL-10; ITG-AvB6; LAG-3 (e.g. relatlimab); Lewis-Y; LG; Ly-6; M-CSF [e.g. PD-0360324 (see US 7,326,414)]; MCSP; mesothelin; MUC1; MUC2; MUC3; MUC4; MUC5AC; MUC5B; MUC7; MUC16; Notch1; notch3; Nectin-4 (e.g. enfortumab vedotin); OX40 [for example PF-04518600 (see US 7,960,515)]; P-cadherin [see for example PF-06671008 (WO 2016/001810)]; PCDHB2; PD-1 [e.g. BCD-100, camrelizumab, cemiplimab, genolimzumab (CBT-501), MEDI0680, nivolumab, pembrolizumab ), RN888 (see WO 2016/092419), sintilimab, spartalizumab, STI-A1110, tislelizumab, TSR-042]; PD-L1 (e.g. atezolizumab, durvalumab, BMS-936559 (MDX-1105) or LY3300054); PDGFRA (e.g. olaratumab); plasma cell antigen; PolySA; PSCA; PSMA; PTK7 [e.g. PF-06647020 (see US 9,409,995)]; Ror1; SAS; SCRx6; SLAMF7 (e.g. elotuzumab); SHH; SIRPa (eg ED9, Effi-DEM); STEAP; TGF-beta; TIGIT; TIM-3; TMPRSS3; TNF-alpha precursor; TROP-2 (e.g. sacituzumab govitecan); TSPAN8; VEGF (e.g. bevacizumab, brolucizumab); VEGFR1 (e.g. ranibizumab); VEGFR2 (e.g. ramucirumab, ranibizumab); Wu-1.

Therapeutic antibodies can have any suitable format. For example, a therapeutic antibody can have any format described herein. In some embodiments, the therapeutic antibody may be a naked antibody. In some embodiments, a therapeutic antibody may be linked to a drug/agent (also known as an “antibody-drug conjugate” (ADC)). In some embodiments, therapeutic antibodies against specific antigens can be incorporated into multispecific antibodies (e.g., bispecific antibodies).

In some embodiments, an antibody directed to an antigen can be conjugated to a drug/agent. Linked antibody-drug molecules are also referred to as “antibody-drug conjugates” (ADCs). The drug/agent can be linked to the antibody directly or indirectly through a linker. Most commonly, the toxic drug is linked to an antibody such that binding of the ADC to the respective antigen promotes death of cells expressing the antigen. For example, ADCs linked to toxic drugs are particularly useful for targeting tumor-associated antigens to promote killing of tumor cells expressing the tumor-associated antigen. In other embodiments, agents that can be linked to the antibody include, for example, immunomodulators (e.g., modulating the activity of immune cells in the vicinity of the ADC), imaging agents (e.g., for imaging the ADC in a subject or biological sample from the subject). It may be an agent that increases antibody serum half-life or biological activity.

Methods for conjugating cytotoxic agents or other therapeutic agents to antibodies are described in various published literature. For example, for the conjugation reaction to occur, there may be chemical modifications in the antibody through lysine side chain amines, or through cysteine sulfhydryl groups activated by reducing interchain disulfide bonds. For example, Tanaka et al., FEBS Letters 579:2092-2096, 2005 and Gentle et al., Bioconjugate Chem. 15:658-663, 2004]. Reactive cysteine residues engineered at specific sites of antibodies for specific drug conjugates with defined stoichiometry have also been described. See, for example, Junutula et al., Nature Biotechnology, 26:925-932, 2008. Glutamine-containing acyl donors made reactive (i.e., capable of forming covalent bonds with acyl donors) by engineering polypeptides in the presence of transglutaminase and amines (e.g., cytotoxic agents containing or attached to reactive amines). Conjugation using tags or endogenous glutamine is also described in WO 2012/059882 and WO2015/015448. In some embodiments, the ADC may have any of the properties and characteristics of the ADC provided in WO 2016/166629, which is incorporated herein by reference for all purposes.

Drugs/agents that can be linked to antibodies in ADC format may include, for example, cytotoxic agents, immunomodulators, imaging agents, therapeutic proteins, biopolymers or oligonucleotides.

Exemplary cytotoxic agents that can be incorporated into the ADC include anthracycline, auristatin, dolastatin, combretastatin, duocarmycin, and pyrrolobenzodia. Zepine dimer, indolino-benzodiazepine dimer, enedyne, geldanamycin, maytansine, puromycin, taxane, vinca alkaloid, campto Includes camptothecin, tubulysin, hemiasterlin, spliceostatin, pladienolide, and stereoisomers, isosteres, analogs or derivatives thereof.

Exemplary immunomodulators that can be incorporated into an ADC include gancyclovier, etanercept, tacrolimus, sirolimus, voclosporin, cyclosporine, rapamycin ( rapamycin), cyclophosphamide, azathioprine, mycophenolgate mofetil, methotrextrate, glucocorticoids and their analogs, cytokines, stem cell growth factors, lymphotoxins, tumor necrosis factor (TNF), Hematopoietic factors, interleukins (e.g. interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, and IL-21), colony-stimulating factors ( interferons (e.g. granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF)), interferons (e.g. interferon-alpha, -beta and -gamma), as "S 1 factor" Contains a designated stem cell growth factor, erythropoietin, thrombopoietin, or a combination thereof.

Exemplary imaging agents that may be included in the ADC include fluorescein, rhodamine, phosphorus lanthanide and its derivatives, or radioactive isotopes coupled to chelators. Examples of fluorophores include, but are not limited to, fluorescein isothiocyanate (FITC) (e.g. 5-FITC), fluorescein amidite (FAM) (e.g. 5-FAM), eosin, carboxyfluorescein. Phosphorus, Erythrosine, Alexa Fluor.RTM. (e.g. Alexa 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 647, 660, 680, 700 or 750 ), carboxytetramethylrhodamine (TAMRA) (e.g. 5,-TAMRA), tetramethylrhodamine (TMR) and sulforhodamine (SR) (e.g. SR101). Examples of chelators include, but are not limited to, 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), 1,4,7-triaza Cyclononane-1,4,7-triacetic acid (NOTA), 1,4,7-triazacyclononane, 1-glutaric acid-4,7-acetic acid (deferoxamine), diethylenetriamine Includes pentaacetic acid (DTPA) and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) (BAPTA).

Exemplary therapeutic proteins that can be included in ADCs include toxins, hormones, enzymes, and growth factors.

Exemplary biocompatible polymers that can be incorporated into ADCs include water-soluble polymers such as polyethylene glycol (PEG) or derivatives thereof, and zwitterion-containing biocompatible polymers (e.g., phosphorylcholine-containing polymers).

Exemplary biocompatible polymers that can be incorporated into ADCs include antisense oligonucleotides.

In some embodiments, antibodies directed to an antigen provided herein can be incorporated into a bispecific antibody molecule. Bispecific antibodies are monoclonal antibodies that have binding specificity for at least two antigens.

In some embodiments, the bispecific antibody comprises a first antibody variable domain and a second antibody variable domain, wherein the first antibody variable domain is capable of activating human immune effector cells by specific binding to CD3 as provided herein. Can be recruited, and the second antibody variable domain can specifically bind to the target antigen. In some embodiments, the antibody has an IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the antibody comprises an immunologically inactive Fc region. In some embodiments, the antibody is a human antibody or humanized antibody.

Target antigens are typically expressed on pathological target cells (eg, cancer cells). Examples of specific target antigens of interest for bispecific antibodies include, but are not limited to, BCMA, EpCAM (epithelial cell adhesion molecule), CCR5 (chemokine receptor type 5), CD19, HER (human epidermal growth factor receptor)-2/neu, HER- 3, HER-4, EGFR (epidermal growth factor receptor), PSMA, CEA, MUC-1 (muchin), MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, CIhCG, Lewis-Y, CD20, CD33, CD30, Gangul Lyoside GD3, 9-O-Acetyl-GD3, GM2, Globo H, Fucosyl GM1, Poly SA, GD2, Carboanhydrase IX (MN/CA IX), CD44v6, Shh (Sonic Hedgehog), Wue- 1, plasma cell antigen, (membrane bound) IgE, MCSP (melanoma chondroitin sulfate proteoglycan), CCR8, TNF-alpha precursor, STEAP, mesothelin, A33 antigen, PSCA (prostate stem cell antigen), Ly-6; Desmoglein 4, E-cadherin neoepitope, fetal acetylcholine receptor, CD25, CA19-9 marker, CA-125 marker and MIS (Müller inhibitor) receptor type II, sTn (sialylated Tn antigen; Tag-72) ), FAP (fibroblast activating antigen), endosialin, EGFRvIII, LG, SAS, PD-L1, CD47, SIRPa, and CD63.

In some embodiments, the bispecific antibody comprises a full-length human antibody, wherein the first antibody variable domain of the bispecific antibody is capable of recruiting the activity of human immune effector cells by specific binding to CD3, and The second antibody variable domain of the monomeric protein can specifically bind to a target antigen (eg CD20, EpCAM or P-cadherin).

Methods for producing bispecific antibodies are known in the art (see, for example, Suresh et al., Methods in Enzymology 121:210, 1986). Typically, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two heavy chains have different specificities (Millstein and Cuello, Nature 305, 537-539, 1983]).

According to one approach for the production of bispecific antibodies, an antibody variable domain with the desired binding specificity (antibody-antigen binding site) is fused to an immunoglobulin constant region sequence. The fusion is preferably performed with an immunoglobulin heavy chain constant region comprising at least a portion of the hinge, CH2 and CH3 regions. It is desirable to have a first heavy chain constant region (CH1) containing the sites required for light chain binding present in one or more fusions. DNA encoding the immunoglobulin heavy chain fusion, and optionally the immunoglobulin light chain, are inserted into separate expression vectors and co-transfected into a suitable host organism. This provides great flexibility in adjusting the ratio of the three polypeptides to one another in embodiments where non-identical ratios of the three polypeptide chains used in the construction provide optimal yield. However, if expression of at least two polypeptide chains in the same ratio results in high yields, or if the ratio has no special meaning, inserting the coding sequences for two or all three of the polypeptide chains in one expression vector may be preferable. possible.

In one approach, a bispecific antibody consists of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure, of which only half of the bispecific molecule is an immunoglobulin light chain, allows separation of the bispecific compound from unwanted immunoglobulin chain combinations. This approach is described in WO 94/04690.

In another approach, the bispecific antibody consists of amino acid modifications in the first hinge region, wherein the substituted/substituted amino acid has an opposite charge to the corresponding amino acid in the second hinge region of another arm. . This approach is described in PCT/US2011/036419 (WO 2011/143545).

In another approach, production of the heteromultimeric or heterodimeric protein of interest (e.g., a bispecific antibody) involves first and second immunoglobulin-like Fc regions (e.g., hinge region and/or CH3 region). ) is enhanced by changing or manipulating the contact between the In this approach, a bispecific antibody may be comprised of a CH3 region, wherein the CH3 region comprises a first CH3 polypeptide and a second CH3 polypeptide, which interact together to form a CH3 contact, wherein one or more of the CH3 contacts within the CH3 contact. Amino acids destabilize homodimer formation and are not electrostatically detrimental to homodimer formation. This approach is described in PCT/US2011/036419 (WO 2011/143545).

In another approach, a bispecific antibody is prepared with a glutamine-containing peptide tag engineered on the antibody to be directed to an epitope (e.g., BCMA) in one arm, and a transglutaminase in the other arm. 2 can be produced using another peptide tag (e.g. a Lys-containing peptide tag or a reactive endogenous Lys) engineered on the antibody to be directed to the epitope. This approach is described in PCT/IB2011/054899 (WO 2012/059882).

In some embodiments, the first and second antibody variable domains of the bispecific antibody comprise amino acid modifications at positions 223, 225, and 228 of the hinge region. Amino acid modifications (e.g., (C223E or C223R), (E225R), and (P228E or P228R)), and amino acid modifications at positions 409 or 368 of the CH3 region of human IgG2 (e.g., K409R or L368E (EU numbering scheme)) Includes.

In some embodiments, the first and second antibody variable domains of the bispecific antibody comprise amino acid modifications at positions 221 and 228 of the hinge region (e.g., (D221R or D221E) and (P228R or P228E)), and and an amino acid modification at positions 409 or 368 of the CH3 region (e.g. K409R or L368E (EU numbering system)).

In some embodiments, the first and second antibody variable domains of the bispecific antibody comprise an amino acid modification at position 228 of the hinge region (e.g., (P228E or P228R)) and an amino acid modification at position 409 or 368 of the CH3 region of a human IgG4. Includes amino acid modifications (e.g. R409 or L368E (EU numbering system)).

In some embodiments, the bispecific antibody may have any of the properties and characteristics of any of the bispecific antibodies provided in WO 2016/166629, which is incorporated herein by reference for all purposes.

Immune modulators include various types of molecules that can stimulate an immune response in an individual, such as pattern recognition receptor (PRR) agonists, immunostimulatory cytokines, and cancer vaccines.

Pattern recognition receptors (PRRs) are receptors expressed by cells of the immune system and recognizing various molecules associated with pathogens and/or cell damage or death. PRRs are involved in both innate and adaptive immune responses. PRR agonists can be used to stimulate an immune response in an individual. Toll-like receptor (TLR), RIG-I-like receptor (RLR), nucleotide-binding oligomerization domain (NOD)-like receptor (NLR), C-type lectin receptor (CLR), and interferon gene (STING) proteins. There are multiple classes of PRR molecules containing stimulatory substances.

The terms “TLR” and “Toll-like receptor” refer to any Toll-like receptor. Toll-like receptors are receptors involved in activating immune responses. TLRs recognize, for example, pathogen-associated molecular patterns (PAMPs) expressed in microorganisms, as well as endogenous damage-associated molecular patterns (DAMPs) released from dead or dying cells.

Molecules that activate TLRs (thereby activating an immune response) are referred to herein as “TLR agonists.” TLR agonists can include, for example, small molecules (e.g., organic molecules with a molecular weight of less than about 1000 daltons), and large molecules (e.g., oligonucleotides and proteins). Some TLR agonists are specific for a single type of TLR (e.g. TLR3 or TLR9), while some TLR agonists activate two or more types of TLR (e.g. both TLR7 and TLR8).

Exemplary TLR agonists provided herein include agonists of TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9.

Exemplary small molecule TLR agonists include, for example, US 4,689,338; 4,929,624; 5,266,575; 5,268,376; 5,346,905; 5,352,784; 5,389,640; 5,446,153; 5,482,936; 5,756,747; 6,110,929; 6,194,425; 6,331,539; 6,376,669; 6,451,810; 6,525,064; 6,541,485; 6,545,016; 6,545,017; 6,573,273; 6,656,938; 6,660,735; 6,660,747; 6,664,260; 6,664,264; 6,664,265; 6,667,312; 6,670,372; 6,677,347; 6,677,348; 6,677,349; 6,683,088; 6,756,382; 6,797,718; 6,818,650; and 7,7091,214; US 2004/0091491, 2004/0176367 and 2006/0100229; and WO 2005/18551, WO 2005/18556, WO 2005/20999, WO 2005/032484, WO 2005/048933, WO 2005/048945, WO 2005/051317, WO 2005/051324, WO 2 005/066169, WO 2005/066170 , WO 2005/066172, WO 2005/076783, WO 2005/079195, WO 2005/094531, WO 2005/123079, WO 2005/123080, WO 2006/009826, WO 2006/009832, WO 2006/026760, WO 2006/028451 , WO 2006/028545, WO 2006/028962, WO 2006/029115, WO 2006/038923, WO 2006/065280, WO 2006/074003, WO 2006/083440, WO 2006/086449, WO 2006/091394, WO 2006/086633 , WO 2006/086634, WO 2006/091567, WO 2006/091568, WO 2006/091647, WO 2006/093514 and WO 2006/098852.

Additional examples of small molecule TLR agonists include certain purine derivatives (such as those described in US 6,376,501 and 6,028,076), certain imidazoquinoline amide derivatives (such as those described in US 6,069,149), certain imidazopyridine derivatives (such as those described in US 6,518,265), Certain benzimidazole derivatives (e.g. those described in US 6,387,938), certain derivatives of 4-aminopyrimidines fused to a 5-membered nitrogen-containing heterocyclic ring (e.g. US 6,376,501; 6,028,076 and 6,329,381; and adenine as described in WO 02/08905) derivatives), certain 3-beta-D-ribofuranosylthiazolo [4,5-d]pyrimidine derivatives (e.g. those described in US 2003/0199461), and certain small molecule immune enhancing compounds (e.g. those described in US 2005/0136065). Includes those listed).

Exemplary large molecule TLR agonists include oligonucleotide sequences. Some TLR agonist oligonucleotide sequences contain cytosine-guanine dinucleotides (CpG) and are described in, for example, US 6,194,388; 6,207,646; 6,239,116; 6,339,068; and 6,406,705. Some CpG-containing oligonucleotides may contain synthetic immunomodulatory structural motifs (such as those described in US Pat. No. 6,426,334 and US Pat. No. 6,476,000). Other TLR agonist nucleotide sequences lack CpG sequences and are described, for example, in WO 00/75304. Another TLR agonist nucleotide sequence is guanosine- and uridine-rich single-stranded RNA (ssRNA) (e.g., those described in Heil et ah, Science, vol. 303, pp. 1526-1529, Mar. 5, 2004). ) includes.

Other TLR agonists include biological molecules such as aminoalkyl glucosaminide phosphate (AGP) and are described in, for example, US 6,113,918; 6,303,347; 6,525,028; and 6,649,172.

Additionally, TLR agonists include inactivated pathogens or fragments thereof, which can activate a number of different types of TLR receptors. Exemplary pathogen-derived TLR agonists include BCG, mycobacterium obuense extract, Talimogene laherparepvec (T-Vec) (derived from HSV-1), and Pexa-Vec (derived from vaccine virus) .

In some embodiments, a TLR agonist may be an agonist antibody that specifically binds to a TLR.

A brief description of various TLRs and TLR agonists is provided below. The following list of TLR agonists for a specific TLR should not be interpreted as indicating that the given TLR agonist necessarily activates only that TLR (e.g., a particular molecule may activate multiple types of TLRs or even multiple classes of PRRs). there is). For example, some molecules provided below as exemplary TLR4 agonists may also be TLR5 agonists.

The terms “TLR1” and “toll-like receptor 1” refer to any form of the TLR1 receptor, and variants, isoforms and species homologs that retain at least a portion of the activities of TLR1. Unless otherwise indicated, such as specifically referring to human TLR1, TLR1 includes native sequence TLR1, the native sequence TLR1 of all mammalian species, such as humans, monkeys and mice. One exemplary human TLR1 is provided under UniProt entry number Q15399.

As used herein, a “TLR1 agonist” means that, when bound to TLR1, (1) stimulates or activates TLR1, (2) enhances, increases, promotes, causes, or prolongs the activity, function, or presence of TLR1; (3) refers to any molecule that enhances, increases, promotes or induces the expression of TLR1. TLR1 agonists useful in any of the therapeutic methods, medicaments and uses of the invention include, for example, bacterial lipoproteins that bind TLR1 and derivatives thereof.

Examples of TLR1 agonists useful in the treatment methods, medicaments and uses of the invention include, for example, bacterial lipoproteins and derivatives thereof, such as SPM-105 (derived from autoclaved mycobacteria), OM-174 (lipid A derivative) , OmpS1 (porin from Salmonella typhi ), OspA (from Borrelia burgdorferi), MALP-2 (mycoplasma macrophage-activating lipopeptide-2kD), STF (soluble tuberculosis factor), CU-T12-9, Diprovocim and lipopeptides derived from cell wall components such as PAM2CSK4, PAM3CSK4 and PAM3Cys.

TLR1 can form heterodimers with TLR2, and therefore many TLR1 agonists are also TLR2 agonists.

The terms “TLR2” and “toll-like receptor 2” refer to any form of the TLR2 receptor, and variants, isoforms and species homologs that retain at least some of the activities of TLR2. Unless otherwise indicated, such as specifically referring to human TLR2, TLR2 includes the native sequence TLR2 of all mammalian species, such as humans, monkeys and mice. One exemplary human TLR2 is provided under Uniprot entry number O60603.

As used herein, a “TLR2 agonist” means that, when bound to TLR2, (1) stimulates or activates TLR2, (2) enhances, increases, promotes, induces, or prolongs the activity, function, or presence of TLR2. , (3) refers to any molecule that enhances, increases, promotes or induces the expression of TLR2. TLR2 agonists useful in any of the therapeutic methods, medicaments and uses of the invention include, for example, bacterial lipoproteins that bind TLR2 and derivatives thereof.

Examples of TLR2 agonists useful in the therapeutic methods, medicaments, and uses of the invention include, for example, bacterial lipoproteins (e.g., diacylated lipoproteins) and derivatives thereof, such as SPM-105 (derived from autoclaved mycobacteria). ), OM-174 (lipid A derivative), OmpS1 (porin from Salmonella typhi), OspA (from Borrelia burgdorferi), MALP-2 (Mycoplasma macrophage-activating lipopeptide-2kD), STF (soluble tuberculosis factor), CU-T12-9, Diprovosim, Amplivant, and lipopeptides derived from cell wall components such as PAM2CSK4, PAM3CSK4 and PAM3Cys.

TLR2 can form heterodimers with TLR1 or TLR6, and therefore many TLR2 agonists are also TLR1 or TLR6 agonists.

The terms “TLR3” and “toll-like receptor 3” refer to any form of the TLR3 receptor, and variants, isoforms and species homologs that retain at least some of the activities of TLR3. Unless otherwise indicated, such as specifically referring to human TLR3, TLR3 includes the native sequence TLR3 of all mammalian species, such as humans, monkeys and mice. One exemplary human TLR3 is provided under Uniprot entry number O15455.

As used herein, a “TLR3 agonist” means that, when bound to TLR3, (1) stimulates or activates TLR3, (2) enhances, increases, promotes, induces, or prolongs the activity, function, or presence of TLR3. , (3) refers to any molecule that enhances, increases, promotes or induces the expression of TLR3. TLR3 agonists useful in any of the treatment methods, medicaments and uses of the invention include, for example, nucleic acid ligands that bind TLR3.

Examples of TLR3 agonists useful in the treatment methods, medicaments, and uses of the invention include TLR3 ligands, such as synthetic dsRNA, polyinosinic acid-polycytidylic acid ["poly(I:C)"] (e.g., available from InvivoGen) available in high molecular weight (HMW) and low molecular weight (LMW) formulations), polyadenylic acid-polyuridylic acid [“poly(A:U)”] (e.g. available from Invogen), polyCLC (Levy et al., Journal of Infectious Diseases, vol. 132, no. 4, pp. 434-439, 1975], Ampligen (Jasani et al., Vaccine, vol. 27, no. 25) -26, pp. 3401-3404, 2009], Hiltonol, Rintatolimod and RGC100 (Naumann et al., Clinical and Developmental Immunology, vol. 2013, article ID 283649) )am.

The terms “TLR4” and “toll-like receptor 4” refer to any form of the TLR4 receptor, and variants, isoforms, and species homologs that retain at least some of the activities of TLR4. Unless otherwise indicated, such as specifically referring to human TLR4, TLR4 includes the native sequence TLR4 of all mammalian species, such as humans, monkeys and mice. One exemplary human TLR4 is provided under Uniprot entry number O00206.

As used herein, a “TLR4 agonist” means that, when bound to TLR4, (1) stimulates or activates TLR4, (2) enhances, increases, promotes, induces, or prolongs the activity, function, or presence of TLR4. , (3) refers to any molecule that enhances, increases, promotes or induces the expression of TLR4. TLR4 agonists useful in any of the therapeutic methods, medicaments and uses of the invention include, for example, bacterial lipopolysaccharide (LPS) and derivatives thereof that bind TLR4.

Examples of TLR4 agonists useful in the treatment methods, medicaments and uses of the invention include, for example, bacterial lipopolysaccharide (LPS) and its derivatives such as B:0111 (Sigma), monophosphoryl lipid A (MPLA) , 3DMPL (3-O-deacylated MPL), GLA-AQ, G100, AS15, ASO2, GSK1572932A (GlaxoSmithKline, UK).

The terms “TLR5” and “toll-like receptor 5” refer to any form of the TLR5 receptor, and variants, isoforms, and species homologs that retain at least some of the activities of TLR5. Unless otherwise indicated, such as specifically referring to human TLR5, TLR5 includes the native sequence TLR5 of all mammalian species, such as humans, monkeys and mice. One exemplary human TLR5 is provided under Uniprot entry number O60602.

As used herein, a “TLR5 agonist” means that, when bound to TLR5, (1) stimulates or activates TLR5, (2) enhances, increases, promotes, induces, or prolongs the activity, function, or presence of TLR5; (3) refers to any molecule that enhances, increases, promotes or induces the expression of TLR5. TLR5 agonists useful in any of the therapeutic methods, medicaments and uses of the invention include, for example, bacterial flagellin and derivatives thereof that bind TLR5.

Examples of TLR5 agonists useful in the therapeutic methods, medicaments and uses of the invention include, for example, bacterial flagellin purified from Bacillus subtilis, flagellin purified from Pseudomonas aeruginosa , Salmonella typhi Flagellin purified from Salmonella typhimurium , and recombinant flagellin (both available from Invogen), Entolimod (CBLB502; a pharmacologically optimized flagellin derivative).

The terms “TLR6” and “toll-like receptor 6” refer to any form of the TLR6 receptor, and variants, isoforms, and species homologs that retain at least some of the activities of TLR6. Unless otherwise indicated, such as specifically referring to human TLR6, TLR6 includes the native sequence TLR6 of all mammalian species, such as humans, monkeys and mice. One exemplary human TLR6 is provided under Uniprot entry number Q9Y2C9.

A “TLR6 agonist” provided herein is one that, when bound to TLR6, (1) stimulates or activates TLR6, (2) enhances, increases, promotes, induces or prolongs the activity, function or presence of TLR6; (3) refers to any molecule that enhances, increases, promotes or induces the expression of TLR6. TLR6 agonists useful in any of the therapeutic methods, medicaments and uses of the invention include, for example, bacterial lipopeptides and derivatives thereof that bind TLR6.

Examples of TLR6 agonists useful in the therapeutic methods, medicaments, and uses of the invention include many of the TLR2 agonists provided above, for example, since TLR2 and TLR6 can form heterodimers. TLR6 can also form heterodimers with TLR4, and TLR6 agonists include the various TLR4 agonists provided above.

The terms “TLR7” and “toll-like receptor 7” refer to any form of the TLR7 receptor, and variants, isoforms, and species homologs that retain at least some of the activities of TLR7. Unless otherwise indicated, such as specifically referring to human TLR7, TLR7 includes the native sequence TLR7 of all mammalian species, such as humans, monkeys and mice. One exemplary human TLR7 is provided under Uniprot entry number Q9NYK1.

As used herein, a “TLR7 agonist” means that, when bound to TLR7, (1) stimulates or activates TLR7, (2) enhances, increases, promotes, induces, or prolongs the activity, function, or presence of TLR7. , (3) refers to any molecule that enhances, increases, promotes or induces the expression of TLR7. TLR7 agonists useful in any of the treatment methods, medicaments and uses of the invention include, for example, nucleic acid ligands that bind TLR7.

Examples of TLR7 agonists useful in the treatment methods, medicaments, and uses of the invention include recombinant single-stranded (“ss”) RNA, imidazoquinoline compounds such as imiquimod (R837), gardiquimod, and regiquimod. resiquimod (R848); Loxoribine (7-allyl-7,8-dihydro-8-oxo-guanosine) and related compounds; 7-thia-8-oxoguanosine, 7-deazaguanosine and related guanosine analogs; ANA975 (Anadys Pharmaceuticals) and related compounds; SM-360320 (Sumimoto); 3M-01, 3M-03, 3M-852 and 3M-S-34240 (3M Pharmaceuticals); GSK2245035 (GlaxoSmithKline; 8-oxoadenine molecule), AZD8848 (AstraZeneca; 8-oxoadenine molecule), MEDI9197 (Medimmune; formerly 3M-052), ssRNA40 and adenosine analogs such as UC -1V150 (Jin et al., Bioorganic Medicinal Chem Lett (2006) 16:4559-4563, compound 4). Many TLR7 agonists are also TLR8 agonists.

The terms “TLR8” and “toll-like receptor 8” refer to any form of the TLR8 receptor, and variants, isoforms, and species homologs that retain at least some of the activities of TLR8. Unless otherwise indicated, such as specifically referring to human TLR8, TLR8 includes the native sequence TLR8 of all mammalian species, such as humans, monkeys and mice. One exemplary human TLR8 is provided under Uniprot entry number Q9NR97.

As used herein, a “TLR8 agonist” means that, when bound to TLR8, (1) stimulates or activates TLR8, (2) enhances, increases, promotes, induces, or prolongs the activity, function, or presence of TLR8; (3) refers to any molecule that enhances, increases, promotes or induces the expression of TLR8. TLR8 agonists useful in any of the treatment methods, medicaments and uses of the invention include, for example, nucleic acid ligands that bind TLR8.

Examples of TLR8 agonists useful in the treatment methods, medicaments and uses of the invention include recombinant single-stranded ssRNA, imiquimod (R837), gardiquimod, resiquimod (R848), 3M-01, 3M-03, 3M-852. and 3M-S-34240 (3M Pharmaceuticals); GSK2245035 (GlaxoSmithKline; 8-oxoadenine molecule), AZD8848 (AstraZeneca; 8-oxoadenine molecule), MEDI9197 (Medimune; formerly 3M-052), Poly-G10, Motolimod and various TLR7s provided above. are agonists (as mentioned above, many TLR7 agonists are also TLR8 agonists).

The terms “TLR9” and “toll-like receptor 9” refer to any form of the TLR9 receptor, and variants, isoforms, and species homologs that retain at least some of the activities of TLR9. Unless otherwise indicated, such as specifically referring to human TLR9, TLR9 includes the native sequence TLR9 of all mammalian species, such as humans, monkeys and mice. One exemplary human TLR9 is provided under Uniprot entry number Q9NR96.

As used herein, a “TLR9 agonist” means that, when bound to TLR9, (1) stimulates or activates TLR9, (2) enhances, increases, promotes, induces, or prolongs the activity, function, or presence of TLR9. , (3) refers to any molecule that enhances, increases, promotes or induces the expression of TLR9. TLR9 agonists useful in any of the treatment methods, medicaments and uses of the invention include, for example, nucleic acid ligands that bind TLR9.

Examples of TLR9 agonists useful in the treatment methods, medicaments and uses of the invention include unmethylated CpG-containing DNA, immunostimulatory oligodeoxynucleotides (ODNs), such as CpG-containing ODNs such as CpG24555, CpG10103, CpG7909 (PF-3512676) /agatolimod), CpG1018, AZD1419, ODN2216, MGN1703, SD-101, 1018ISS and CMP-001. TLR9 agonists also include the nucleotide sequence containing the synthetic cytosine-phosphate-2'-deoxy-7-deazaguanosine dinucleotide (CpR) (Hybridon, Inc.), dSLIM-30L1, and immunotherapy. Contains a globulin-DNA complex. Exemplary TLR9 agonists include WO 2003/015711, WO 2004/016805, WO 2009/022215, PCT/US95/01570, PCT/US97/19791, and US 8,552,165, 6,194,388 and 6,239,116 (each of which is incorporated herein by reference for all purposes) note on circle (included) is disclosed.

RLRs include a variety of cytosolic PRRs that, for example, detect dsRNA. Examples of RLRs include, for example, retinoic acid-inducible gene I (RIG-I), melanoma differentiation associated gene 5 (MDA-5), and Laboratory of Genetics and Physiology 2 (LGP2).

As used herein, an “RLR agonist” means that, when bound to an RLR, (1) stimulates or activates an RLR, (2) enhances, increases, facilitates, induces, or prolongs the activity, function, or presence of an RLR. , (3) refers to any molecule that enhances, increases, promotes or induces the expression of RLR. RLR agonists useful in any of the therapeutic methods, medicaments, and uses of the invention include, for example, nucleic acids and derivatives thereof that bind RLR, and functional monoclonal antibodies (mAbs) that specifically bind RLR.

Examples of RLR agonists useful in the therapeutic methods, medicaments, and uses of the invention include, for example, short double-stranded RNA with an uncapped 5' triphosphate (RIG-I agonist); Poly I:C (MDA-5 agonist) and BO-112 (MDA-A agonist).

NLRs include a variety of PRRs that detect, for example, damage associated molecular pattern (DAMP) molecules. NLR includes subfamilies NLRA-A, NLRB-B, NLRC-C, and NLRP-P. Examples of NLRs include, for example, NOD1, NOD2, NAIP, NLRC4, and NLRP3.

As used herein, an “NLR agonist” means that, when bound to an NLR, (1) stimulates or activates an NLR, (2) enhances, increases, promotes, induces, or prolongs the activity, function, or presence of an NLR. , (3) refers to any molecule that enhances, increases, promotes or induces the expression of NLR. NLR agonists useful in any of the treatment methods, medicaments and uses of the present invention include, for example, DAMPs that bind to NLR and their derivatives, and functional monoclonal antibodies (mAbs) that specifically bind to NLRs.

An example of a NLR agonist useful in the treatment methods, medicaments and uses of the invention is, for example, liposomal muramyl tripeptide/mifamurtide (NOD2 agonist).

CLRs include a variety of PRRs that detect, for example, carbohydrates and glycoproteins. CLRs include both transmembrane CLRs and secreted CLRs. Examples of CLRs include, for example, DEC-205/CD205, macrophage mannose receptor (MMR), Dectin-1, Dectin-2, mincle, DC-SIGN, DNGR-1, and mannose binding lectin (MBL). ) includes.

As used herein, a “CLR agonist” means that, when bound to a CLR, (1) stimulates or activates a CLR, (2) enhances, increases, facilitates, induces, or prolongs the activity, function, or presence of a CLR. , (3) refers to any molecule that enhances, increases, promotes or induces the expression of CLR. CLR agonists useful in any of the therapeutic methods, medicaments and uses of the invention include, for example, carbohydrates and derivatives thereof that bind to the CLR, and functional monoclonal antibodies (mAbs) that specifically bind to the CLR.

Examples of CLR agonists useful in the therapeutic methods, medicaments and uses of the present invention include, for example, MD-fraction (purified soluble beta-glycan extract from Grifola frondosa ) and imprime PGG ( It is a beta 1,3/1,6-glucan PAMP) derived from yeast.

STING proteins act as both cytosolic DNA sensors and adapter proteins in type 1 interferon signaling. The terms “STING” and “stimulator of the interferon gene” refer to any form of the STING protein, and variants, isoforms, and species homologs that retain at least a portion of STING activity. Unless otherwise indicated, such as specifically referring to human STING, STING includes the native sequence STING of all mammalian species, such as humans, monkeys and mice. One exemplary human TLR9 is provided under Uniprot entry number Q86WV6. STING is also known as TMEM173.

As used herein, a “STING agonist” means that, when bound to TLR9, (1) stimulates or activates STING, (2) enhances, increases, promotes, induces, or prolongs the activity, function, or presence of STING. , (3) refers to any molecule that enhances, increases, promotes or induces the expression of STING. STING agonists useful in any of the therapeutic methods, medicaments and uses of the invention include, for example, nucleic acid ligands that bind STING.

Examples of STING agonists useful in the therapeutic methods, medicaments, and uses of the invention include various immunostimulatory nucleic acids, such as synthetic double-stranded DNA, cyclic di-GMP, cyclic-GMP-AMP (cGAMP), synthetic cyclic dinucleotide (CDN) ), such as MK-1454 and ADU-S100 (MIW815), and small molecules such as P0-424.

Other PRRs include, for example, DNA-dependent activator of IFN-regulatory factors (DAI) and Absent in Melanoma 2 (AIM2).

Immunostimulatory cytokines include a variety of signaling proteins that stimulate immune responses, such as interferons, interleukins, and hematopoietic growth factors.

Exemplary immune stimulatory cytokines include GM-CSF, G-CSF, IFN-alpha, IFN-gamma; Includes IL-2 (e.g. denileukin difitox), IL-6, IL-7, IL-11, IL-12, IL-15, IL-18, IL-21 and TNF-alpha. do.

Immune stimulatory cytokines may have any suitable format. In some embodiments, the immunostimulatory cytokine may be a recombinant version of a wild-type cytokine. In some embodiments, the immunostimulatory cytokine may be a mutein with one or more amino acid changes compared to the corresponding wild-type cytokine. In some embodiments, immunostimulatory cytokines can be incorporated into a chimeric protein (e.g., an antibody) containing a cytokine and one or more other functional proteins. In some embodiments, the immunostimulatory cytokine may be covalently linked to a drug/agent (e.g., any of the drugs/agent described herein as a possible ADC component).

Cancer vaccines include a variety of compositions that contain tumor-associated antigens (or can be used to produce tumor-associated antigens in an individual), and thus can be used to induce an immune response in an individual, which contain tumor-associated antigens. directed to tumor cells.

Exemplary agents that can be included in a cancer vaccine include attenuated cancerous cells, tumor antigens, antigen presenting cells, such as dendritic cells pulsed with nucleic acids encoding tumor derived antigens or tumor associated antigens. In some embodiments, the cancer vaccine can be produced by the patient's own cancer cells. In some embodiments, a cancer vaccine can be produced by biological material that is not derived from the patient's own cancer cells.

Cancer vaccines include, for example, sipuleucel-T and talimogene laherparepvec (T-VEC).

Immune cell therapy involves treating patients with immune cells that can target cancer cells. Immune cell therapies include, for example, tumor-infiltrating lymphocytes (TILs) and chimeric antigen receptor T cells (CAR-T cells).

Examples of chemotherapeutic agents include: alkylating agents such as thiotepa and cyclophosphamide; Alkyl sulfonates such as busulfan, improsulfan and piposulfan; Aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethyleneimide and methylamelamine, such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamamine; Acetogenins (especially bullatacin and bullatacinone); camptothecin (including the synthetic analogue topotecan); bryostatin; calllistatin; CC-1065 (including its azozelesin, carzelesin and bizelesin synthetic analogs); cryptophycin (especially cryptophycin 1 and cryptophycin 8); dolastatin; Duocarmycin (including synthetic analogs, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; sarcodictyin; spongestatin; Nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechloreta Minoxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosurea, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; Antibiotics, such as endoyne antibiotics (e.g. calicheamicin, especially calicheamicin gamma1I and calicheamicin phiI1 (e.g. Agnew, Chem. Intl. Ed. Engl., 33:183- 186 (1994); dynemicin (including dynemicin A); bisphosphonates such as clodronate; esperamicin; and neocarzinostatin Chromophore and related pigment protein endoyne antibiotics chromophore, alacinomysin, actinomycin, authramycin, azaserine, bleomycin, cactino cactinomycin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin ), 6-diazo-5-oxo-L-norleucine, doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), pegylated Liposomal doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, such as mitomycin C, mycophenolic acid, Nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin (streptonigrin), streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); Folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; Purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; Pyrimidine analogs such as ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine ), encocitabine, floxuridine; Androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; Anti-adrenergics such as aminoglutethimide, mitotane, trilostane; Folic acid supplements such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatrexate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone; Podophyllic acid; 2-ethylhydrazide; procarbazine; razoxane; rhizoxin; sizofuran; Spirogermanium; tenuazonic acid; triaziquone; 2, 2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethanes; Vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside )("Ara-C"); cyclophosphamide; thiotepa; taxoids such as paclitaxel and docetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs , such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine ); Novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomera Inhibitors RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Also included are: Antihormonal agents that act to modulate or inhibit the action of hormones on the body, such as antiestrogens and selective estrogen receptor modulators (SERMs), such as tamoxifen, raloxifene, droloxifene, 4-hyde Roxitamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); Aromatase inhibitors, which inhibit the enzyme aromatase that regulates estrogen production in the adrenal glands, such as 4(5)-imidazole, aminoglutethimide, megestrol acetate, exemestane, formestane ( formestane, fadrozole, vorozole, letrozole and anastrozole; and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; KRAS inhibitors; MCT4 inhibitor; MAT2a inhibitor; Tyrosine kinase inhibitors such as sunitinib, axitinib; alk/c-Met/ROS inhibitors such as crizotinib, lorlatinib; mTOR inhibitors such as temsirolimus, gedatolisib; src/abl inhibitors such as bosutinib; Cyclin dependent kinase (CDK) inhibitors such as palbociclib, PF-06873600; erb inhibitors such as dacomitinib; PARP inhibitors such as talazoparib; SMO inhibitors such as glasdegib, PF-5274857; EGFR T790M inhibitors such as PF-06747775; EZH2 inhibitors such as PF-06821497; PRMT5 inhibitors such as PF-06939999; TGFRβr1 inhibitors such as PF-06952229; A pharmaceutically acceptable salt, acid or derivative of any of the foregoing. In specific embodiments, these additional therapeutic agents include bevacizumab, cetuximab, sirolimus, panitumumab, 5-fluorouracil (5-FU), capecitabine, tivozanib, irinotecan, oxaplatin, Cisplatin, trifluridine, tipiracil, leucovorin, gemcitabine, regorafinib, or erlotinib hydrochloride.

In some embodiments, the antibody is administered with one or more other therapeutic agents targeting an immune checkpoint modulator or co-stimulator, such as, but not limited to, CTLA-4, LAG-3, B7-H3, B7-H4, B7-DC (PD-L2 ), B7-H5, B7-H6, B7-H8, B7-H2, B7-1, B7-2, ICOS, ICOS-L, TIGIT, CD2, CD47, CD80, CD86, CD48, CD58, CD226, CD155, CD112, LAIR1, 2B4, BTLA, CD160, TIM1, TIM-3, TIM4, VISTA(PD-H1), OX40, OX40L, GITR, GITRL, CD70, CD27, 4-1BB, 4-BBL, DR3, TL1A, CD40 , CD40L, CD30, CD30L, LIGHT, HVEM, SLAM (SLAMF1, CD150), SLAMF2 (CD48), SLAMF3 (CD229), SLAMF4 (2B4, CD244), SLAMF5 (CD84), SLAMF6 (NTB-A), SLAMCF7 (CS1) ), SLAMF8 (BLAME), SLAMF9 (CD2F), CD28, CEACAM1 (CD66a), CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM7, CEACAM8, CEACAM1-3AS CEACAM3C2, CEACAM1-15, PSG1-11, CEACAM1-4C1, CEACAM1- Agents targeting 4S, CEACAM1-4L, IDO, TDO, CCR2, CD39-CD73-adenosine pathway (A2AR), BTKs, TIKs, CXCR2, CXCR4, CCR4, CCR8, CCR5, CSF-1, or innate immune response modulators are used together

In some embodiments, the antibody is, for example, an anti-CTLA-4 antagonist antibody, such as ipilimumab; anti-LAG-3 antagonist antibodies such as BMS-986016 and IMP701; anti-TIM-3 antagonist antibody; anti-B7-H3 antagonist antibodies such as MGA271; anti-VISTA antagonist antibody; anti-TIGIT antagonist antibody; antibodies; anti-CD80 antibody; anti-CD86 antibody; anti-B7-H4 antagonist antibody; anti-ICOS agonist antibody; anti-CD28 agonist antibody; It is used in combination with innate immune response modulators (e.g. TLRs, KIR, NKG2A) and IDO inhibitors.

In some embodiments, the antibody is used in conjunction with an OX40 agonist, such as an anti-OX-40 agonist antibody. In some embodiments, the antibody is used in combination with a GITR agonist, such as an anti-GITR agonist antibody such as, but not limited to, TRX518. In some embodiments, the antibody is used in combination with an IDO inhibitor. In some embodiments, the antibody is used in conjunction with a cytokine therapeutic such as, but not limited to, IL-15, CSF-1, MCSF-1, etc.

In some embodiments, the antibody is used in combination with one or more other therapeutic antibodies, such as, but not limited to, antibodies targeting CD19, CD22, CD40, CD52, or CCR4.

In certain embodiments, the antibody composition can be combined with one or more additional agents such as bevacizumab, cetuximab, sirolimus, panitumumab, 5-fluorouracil (5-FU), capecitabine, tivozanib, irinotecan, Includes oxaplatin, cisplatin, trifluridine, tipiracil, leucovorin, gemcitabine, and erlotinib hydrochloride.

In some embodiments, antibody therapy may be co-administered with other agent treatments, or may precede or follow other agent treatments by intervals ranging from minutes to weeks. In embodiments where the other agents and/or proteins or polynucleotides are administered separately, it is generally ensured that a significant period of time has not expired between each delivery so that the agents and compositions of the invention are still combined to the benefit of the individual. effect can be exercised. In such cases, it is contemplated that both modalities may be administered within about 12 to 24 hours of each other, more preferably within about 6 to 12 hours of each other. In some cases, it may be advisable to extend the dosing period significantly, from a few days (2, 3, 4, 5, 6 or 7) to a few weeks (1, 2, 3, 4, 5, 6, or 7) between each dosing. 7 or 8) passes.

In some embodiments, the antibodies of the invention are administered in a treatment regimen further comprising a conventional therapy selected from the group consisting of surgery, radiation therapy, chemotherapy, targeted therapy, immunotherapy, hormonal therapy, angiogenesis inhibition, and palliative therapy. It is used together.

The composition used in the present invention is a lyophilized pharmaceutically acceptable carrier, excipient, or stabilizer (Remington: The Science and practice of Pharmacy 21st Ed., 2005, Lippincott Williams and Wilkins, Ed. K. E. Hoover]). It may be additionally included in the form of a formulation or aqueous solution. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations and include buffers such as phosphate, citrate, and other organic acids; Antioxidants such as ascorbic acid and methionine; Preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclo Hexanol; 3-pentanol; and m-cresol; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine , glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates such as glucose, mannose or dextran; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salts- Forming counterions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or nonionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Pharmacology Commercially acceptable excipients are further described herein.

In one aspect, the invention provides a method for producing an antibody disclosed herein, comprising culturing a host cell under conditions that result in production of the antibody disclosed herein and purifying the antibody or bispecific antibody from the culture. do.

In another aspect, the invention provides the use of an antibody, pharmaceutical composition, polynucleotide, vector or host cell disclosed herein in the manufacture of a medicament for the detection, diagnosis and/or treatment of tumor antigen associated disorders.

Diagnostic use

In one aspect, the CD3 antibodies of the invention can be used to detect and/or measure CD3 or CD3-expressing cells in a sample, for example, for diagnostic purposes. For example, CD3 antibodies can be used to diagnose diseases or disorders characterized by abnormal expression of CD3 (e.g., overexpression, underexpression, lack of expression, etc.). Exemplary diagnostic assays for CD3 may include, for example, contacting a sample obtained from an individual with a CD3 antibody of the invention, wherein the CD3 antibody is labeled with a detectable label or reporter molecule. Alternatively, unlabeled CD3 antibodies can be used in diagnostic applications in combination with a secondary antibody that is itself detectably labeled. Detectable labels or reporter molecules include radioactive isotopes such as ¹⁴ C, ³ H, ¹²⁵ I, ³² P or ³⁵ S; Fluorescent or chemiluminescent moieties such as fluorescein isothiocyanate or rhodamine; or an enzyme such as alkaline phosphatase, beta-galactosidase, horseradish peroxidase or luciferase. Specific exemplary assays that can be used to detect or measure CD3 in a sample include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence activated cell sorting (FACS), and the like. Samples that can be used in a CD3 diagnostic assay according to the present invention include any tissue or fluid sample obtainable from a patient that contains detectable amounts of CD3 protein or fragments thereof under normal or pathological conditions. Typically, the level of CD3 in a particular sample obtained from a healthy patient (e.g., a patient not suffering from a disease or condition associated with abnormal CD3 levels or activity) will be measured to establish a baseline or reference level of CD3. These baseline levels of CD3 can then be compared to CD3 levels measured in samples obtained from individuals suspected of having a CD3-related disease or condition.

In another aspect, a method for detecting, diagnosing, and/or monitoring a disease associated with tumor antigen expression is provided. For example, the multispecific antibodies described herein can be labeled with detectable moieties such as imaging agents and enzyme-substrate labels. Additionally, the antibodies described herein can be used in in vitro diagnostic assays, such as in vivo imaging (e.g. PET or SPECT), or staining reagents.

kit

A further aspect of the invention is a kit comprising the antibodies disclosed herein above, and instructions for use in accordance with any of the methods described herein. Generally, these instructions include instructions for administration of the antibody for the therapeutic treatment described above. These kits include any of the pharmaceutical compositions disclosed herein. Pharmaceutical compositions and other reagents may be present in the kit in any conventional form, such as solutions or powders.

In another aspect, the kit further includes in one or more containers one or more other prophylactic or therapeutic agents useful in the treatment of cancer. In one embodiment, the other prophylactic or therapeutic agent is a chemotherapeutic agent. In another aspect, the prophylactic or therapeutic agent is a biotherapeutic agent or a hormonal agent.

The various aspects of the pharmaceutical composition, prophylactic or therapeutic agent of the invention are preferably tested for the desired therapeutic activity in in vitro cell culture systems and in animal model organisms, such as rodent animal model systems, prior to use in humans.

The toxicity and potency of the prophylactic and/or therapeutic protocol of the invention can be determined by, for example, LD ₅₀ (lethal dose for 50% of the population) and ED ₅₀ (therapeutically effective dose for 50% of the population). Determination can be made in cell culture or laboratory animals by standard pharmaceutical procedures. The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD ₅₀ /ED ₅₀ . Prophylactic and/or therapeutic agents that exhibit a large therapeutic index are preferred.

Additionally, any assay known to those skilled in the art can be used to evaluate the prophylactic and/or therapeutic utility of the therapies or combination therapies disclosed herein for the treatment or prevention of cancer.

Instructions for use of the bispecific antibodies described herein generally include information regarding dosage, schedule of administration, and route of administration for the intended treatment. The container may be a unit dose, bulk package (eg, multi-dose package), or subunit dose. The instructions supplied with the kits of the invention are typically written instructions on a label or package insert (e.g., a sheet of paper included in the kit), but are also machine-readable instructions (e.g., instructions on a magnetic or optical storage disk). ) is also acceptable.

Kits of the invention are presented in suitable packaging. Suitable packaging materials for each pharmaceutical composition and other contained reagents, such as buffers, balanced salt solutions, etc., for administering the pharmaceutical composition to a subject include, but are not limited to, vials, amplifiers, tubes, bottles, jars, flexibles, etc. Includes flexible packaging materials (e.g., sealed Mylar or plastic bags). Also contemplated are packaging materials for use in combination with certain devices, such as inhalers, nasal administration devices (e.g., atomizers), or infusion devices, such as minipumps. The kit may have a sterile access port (for example, the container may be an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle). The container may also have a sterile access port (for example, the container may be an intravenous solution bag or vial with a stopper through which a hypodermic needle can penetrate). At least one active agent in the composition is an antibody. The container may additionally contain a second pharmaceutically active agent.

Typically, a kit includes a container and a label or package insert on or associated with the container.

biological deposit

Representative material of the invention was deposited with the American Type Culture Collection (ATCC) (10801 University Blvd., Manassas, VA 20110-2209, USA) on February 13, 2018. Vector GUCY2C-1608 Chain A (VH-hole SEQ ID NO: 94), with ATCC accession number PTA-124943, is a bispecific variant comprising a polynucleotide (SEQ ID NO: 70) encoding the CD3 antibody light chain variable region (SEQ ID NO: 3). Contains a DNA insert encoding the VH-hole chain of the red antibody GUCY2C-1608; Vector GUCY2C-1608 chain B (VL-knob SEQ ID NO: 93) with ATCC accession number PTA-124944 is a bispecific protein comprising a polynucleotide (SEQ ID NO: 71) encoding the CD3 antibody heavy chain variable region (SEQ ID NO: 4). It contains a DNA insert encoding the VL-knob chain of the red antibody GUCY2C-1608.

The deposit was made under the provisions of the international recognition of deposits of microorganisms for the purposes of patent proceedings and the provisions under such treaty (Budapest Treaty). This ensures the maintenance of viable cultures of the deposit for 30 years from the date of deposit. Deposits will be available from ATCC under the provisions of the Budapest Treaty, pursuant to an agreement between Pfizer, Inc. and ATCC, at the time of registration of the relevant U.S. patent or in the filing of any U.S. or foreign patent application. Upon disclosure (whichever is sooner), ensure permanent, unrestricted availability of the progeny of the deposited culture to the public, and comply with 35 U.S.C. Section 122 and the rules of the Commissioner of the United States Patent and Trademark Office thereunder (including 37 C.F.R. Section 1.14 with specific reference to 886 OG 638) ensure availability to descendants of those qualified by the Commissioner of the United States Patent and Trademark Office.

The assignee hereby agrees that if a culture of the deposited material dies, is lost or is destroyed when cultured under suitable conditions, it will promptly replace that material with another identical one upon notice. The availability of deposited materials should not be construed as a license to practice the invention contrary to the rights secured under the authority of any government under the Patent Act.

The following examples of specific aspects for carrying out the invention are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way.

Example 1: Production of soluble CD3ed

A protein referred to as CD3ed, which represents the extracellular domains of the CD3 epsilon and delta subunits fused side by side via a short linker (SEQ ID NO: 39), was produced using standard molecular biology, protein expression and purification techniques. Plasmids containing genes encoding the amino acids shown in Table 9 (SEQ ID NO: 40) were transiently transfected into HEK293 cells in a 20 L culture volume using PEI as a transfection agent. After culturing at 37°C for 7 days, 20 L of the harvest was batch-bound overnight in 50 mL of His60 resin (equilibrated with PBS). The combined resin was then washed with PBS and then with 10 mM imidazole-containing PBS buffer (10 to 15 CV). After washing, proteins were eluted with 200 mM imidazole-containing phosphate buffer. The eluting fractions were then analyzed by SDS gel. Fractions containing pure protein were pooled together and concentrated using 3 kDa Amicon ultra centricons. 5% glycerol was maintained in the samples during concentration to keep the proteins stable. The His60 pool was further loaded onto a size exclusion Superdex200 column. Fragments containing the CD3 epsilon/delta heterodimer were analyzed by SDS-PAGE, and relevant fractions were pooled and 0.22 μm filtered. Table 9 shows the sequences of CD3 epsilon/delta subunits fused in tandem, used as antigens for optimization work.

[Table 9]

Example 2: Framework optimization of CD3 antibody H2B4

The lead CD3e (CD3 epsilon) antibody H2B4 showed low thermal stability when used in the bispecific diabody-Fc format. To improve stability, the light chain framework was changed from VK4 to VK1 (DPK9) while the heavy chain framework remained VH3 (DP54). VL CDR1 (SEQ ID NO: 26), VL CDR2 (SEQ ID NO: 27), and VL CDR3 (SEQ ID NO: 28) were transplanted into the DPK9 scaffold and CDR, VH CDR1 (SEQ ID NO: 13), VH CDR2 (SEQ ID NO: 16), and VH CDR3 (SEQ ID NO: It was expressed in combination with a heavy chain containing SEQ ID NO: 18). In addition to grafting CDRs into the scaffold, specific reversion mutations were also tested. The back mutations introduced into both the heavy and light chain backbones are shown in Table 10, indicating the residue numbers at which the back mutations were performed.

cDNA containing the human acceptor framework, VH3 (heavy chain) and VK1 (light chain), and the associated CDR donor sequences were synthesized. The synthesized cDNA product was subcloned and fused in frame with human IgG1 constant region (heavy chain) and human kappa (light chain) in a mammalian expression vector. To distinguish them from the H2B4 antibody, these variants are referred to as 2B5, and all optimized molecules are hereafter referred to as 2B5 and its variants. All 2B5 mutants were analyzed for competitive binding with Jurkat cells endogenously expressing CD3e.

Competitive FACS was performed to test whether humanized 2B5 retained its binding epitope on the cell surface. Before competition FACS experiments, chimeric cH2B4 was biotinylated. The EC ₅₀ of binding of biotinylated cH2B4 to Jurkat cells was determined by direct binding using FACS and was determined to be 0.8 nM. In competitive FACS, 3-fold serially diluted antibody variants were mixed with a constant amount of biotin-cH2B4 representing EC ₅₀ concentration and 1.0+E05 cells/well of Jurkat cells. The mixture was incubated on ice for 1 hour and then washed 3x with FACS buffer. The secondary antibody, phycoethrin (PE) conjugated streptavidin (PE-SA), was added to the cells and incubated on ice for 30 min. Median fluorescence intensity (MFI) was then analyzed by FACS on a FACS CANTO™ (BD Biosciences). Y-axis calculation: % binding to Max = [MFI (serially diluted Ab wells) - MFI (2nd PE-SA)] / [MFI max (Jurkat cells with biotin-cH2B4 only) - MFI (2nd PE-SA)]. Table 10 shows the results of the IC ₅₀ values of the competition assay when mutations were introduced into the CD3 antibody H2B4 to optimize the framework. Based on these results, H2B4 1.0/1.0T showed binding characteristics similar to H2B4 and was designated as CD3 antibody binding domain 2B5.

[Table 10]

Example 3: Production and characterization of bispecific diabody-Fc molecules using H2B4 and 2B5 sequences

Bispecific diabody-Fc molecules were generated using standard expression and purification techniques known in the art using CD3 antibody 2B5 or H2B4 (Tables 2 and 3) and GUCY2c antibody sequences in one of the arrangements shown in Figure 1 (schematic). produced. The bispecific antibodies produced (Table 7; SEQ ID NOs: 36 to 38) were tested for stability using differential scanning calorimetry.

Example 4: Evaluation of the stability of bispecific antibodies using differential scanning calorimetry

Proteins were diluted to 0.6 mg/mL in phosphate buffered saline (PBS) solution in a volume of 400 μL. PBS was used in reference cells as a buffer blank. PBS _contained : 137mM NaCl, 2.7mM KCl, _{8.1mM Na 2HPO4 and 1.47mMKH2PO4} , pH 7.2. Samples were dispensed into the sample tray of a MicroCal VP-Capillary DSC using an autosampler (Malvern Instruments Ltd, Malvern, UK). Samples were equilibrated at 10°C for 5 minutes and then scanned at 100°C/hour up to 110°C. A filtration time of 16 seconds was chosen. Raw data were baseline corrected and protein concentrations were normalized. Data were fit to the MN2-State model with the appropriate number of transitions using Origin software 7.0 (OriginLab Corporation, Northampton, MA, USA). As shown in Figure 2, the GUCY2c-2B5 bispecific antibody showed an improved first melting transition of 5°C compared to the GUCY2c-H2B4 bispecific antibody. This suggests that the bispecific antibody containing 2B5 shows better stability.

The two bispecific antibodies were confirmed for binding to T cells using FACS using standard procedures known in the art, and as shown in Figure 3, both bispecific antibodies showed similar binding to T cells. binding, suggesting that binding is preserved upon conversion from H2B4 to 2B5.

Additionally, both H2B4 and 2B5 bispecific antibodies were tested in a cytotoxicity assay to assess T cell redirecting activity. As shown in Figure 4, both bispecific antibodies showed similar cytotoxicity, suggesting that they maintained the same activity as H2B4.

Example 5: Rational mutagenesis of 2B5 based on structure

The CD3 antibody H2B4 binding domain was polyreactive as assessed by binding to DNA and insulin, showed a tendency to self-associate as suggested by high AC-SINS scores, and CDR-H2 when expressed in CHO host cells. had a clipping tendency at the R53 position. To reduce polyreactivity, self-association tendency, and clipping of CDRH2, mutations in the CDR of 2B5 were engineered to reduce these disadvantages while maintaining binding affinity and stability. For this purpose, all predictions were performed using the x-ray crystal structure of CD3 antibody H2B4, which represents the binding epitope and was obtained in complex with a peptide derived from the N-terminus of the CD3 epsilon subunit. It is well known in the art that excessive positive charge and/or hydrophobicity can play a major role in polyreactivity. The electrostatic surface of the H2B4 Fv region was determined from the crystal structure using the DelPhi Poisson Boltzmann calculator (Discovery Studio 4.0 ¹ ). In Figure 5, excessively positively charged patches were observed that were not destroyed by negatively charged patches. This suggests a correlation with high polyreactivity. Additionally, excessive hydrophobic patches (Figure 6) were determined using the spatial aggregation propensity (SAP) tool (Discovery Studio1). As shown in Figure 6, the hydrophobic patches appear to be small and sparse. Based on these observations, the focus was on the reduction of positive charge by removal of positive charge or addition of negative charge to destroy the patch.

To determine suitable sites for mutagenesis, computational prediction of mutations for both affinity changes and stability changes was performed using Discovery Studio and FoldX. Acceptable mutations are those that are not predicted to have a ΔΔG greater than 1 kcal/mol in both the Discovery Studio and FoldX methods. From these predictions, a set of predicted residues were identified that would have minimal impact on binding and stability, while still disrupting the change patch. Additionally, when possible, hydrophilic mutations were preferred to avoid increasing the hydrophobic surface. A list of possible CDR mutations introduced into CD3 antibody 2B5 to optimize biophysical properties is shown in Table 11. This includes mutations at each position that are tolerated and predicted to reduce polyreactivity and clipping.

[Table 11]

Additionally, multiple sets (combinations) of mutations were designed for the heavy and light chains. These are R52Q/R52bQ/R53Q, R52Q/R52bQ/R53Q/R96Q, R52bQ/R53Q/R96Q and R52Q/R52bQ/R96Q for the heavy chain, and R29Q/K30Q/R54L/V27eE, R29Q_R54L_V27eE, R29Q_K for the light chain. Includes 30Q_R54L.

Example 6: Characterization of mutants

All mutants were produced as IgG molecules using standard expression and purification techniques well known in the art and tested for binding to CD3ed, polyreactivity (binding to DNA and insulin) and AC-SINS.

Binding assay to evaluate variants

An Octet assay was designed and performed to test association and dissociation rates at 10 μg/mL soluble human CD3ed antigen (SEQ ID NO: 40). Anti-CD3 antibody was captured using an anti-human IgG tip for 120 s at a concentration of 10 μg/mL, followed by 30 s in PBS, and soluble human CD3ed antigen at a concentration of 10 μg/mL for 120 s. Soaked and finally dissociated for 300 seconds. Sensorgrams were examined to determine whether the antigen bound to the antibody, and k _d (1/s) and k _d error were calculated using Octet software. Twenty-six plausible mutations retained binding to hCD3ed antigen. The kinetics of binding to soluble CD3 for the 26 CD3 antibody variants that remained bound to antigen were then tested by SPR using Biacore analysis. CD3 antibody was captured on anti-human Fc, and soluble CD3 antigen at concentrations of 0, 3.7, 11.1, 33.3, and 100 nM were injected into the captured antibody to determine binding kinetics. Twenty-three CD3 antibodies bound to soluble CD3 antigen with affinities similar to those of the CDR-grafted parent antibodies.

DNA and insulin ELISA to measure polyreactivity

384-well ELISA plates (Nunc Maxisorp) were incubated with DNA (10 μg/mL) (Sigma-Aldrich, D1626) and insulin (5 μg/mL) (Sigma-Aldrich) in PBS (pH 7.5) overnight at 4°C. Aldrich, I9278-5mL). An ELISA modified from the assay described by Tiller et al (17) was performed on a PerkinElmer Janus automated workstation liquid handling robot. Wells were washed with water, blocked with 50 μL of polyreactive ELISA buffer (PEB; PBS containing 0.05% Tween-20 and 1 mM EDTA) for 1 h at room temperature, and rinsed with water three times. Serially diluted mAb in 25 μL was added to the wells in quadruplicate experiments and incubated at room temperature for 1 hour. The plate was washed three times with water and 25 μL of 10 ng/mL goat anti-human IgG (Face fragment specific) conjugated to horseradish peroxidase (Face fragment specific) (Jackson ImmunoResearch, 109-035-008) was added to each well. was added to. Plates were incubated at room temperature for 1 hour, washed three times with 80 μL of water, and 25 μL of TMB substrate (Sigma Aldrich, T-0440) was added to each well. The reaction was stopped after 7 minutes by adding 25 μL of 0.18 M ortho-phosphoric acid to each well and the absorbance was read at 450 nm. DNA- and insulin-binding scores were calculated as the ratio of the ELISA signal of the antibody at 10 μg/mL to the signal of the well containing buffer instead of the primary antibody. A score of less than 8 is considered ideal and indicates low polyreactivity.

AC-SINS analysis measuring self-association tendency

The AC-SINS (Affinity Capture Self-Interaction Nanoparticle Spectroscopy) assay is standardized in a 384-well format on a Perkin-Elmer Janus liquid handling robot. 20 nm gold nanoparticles (Ted Pella, Inc. #15705) were buffer exchanged with 20 mM sodium acetate (pH 4.3) and diluted to 0.4 mg/mL, 80% goat anti-human. with a mixture of Fc (Jackson ImmunoResearch Laboratories, Inc. # 109-005-098) and 20% non-specific goat polyclonal antibody (Jackson ImmunoResearch Laboratories, Inc. # 005-000-003). Coated. After 1 hour of incubation at room temperature, unoccupied sites on the gold nanoparticles were blocked with thiolated polyethylene glycol (2 kD). The coated nanoparticles were then concentrated 10-fold using a syringe filter, and 10 μL was added to 100 μL of mAb at 0.05 mg/mL in PBS (pH 7.2). The coated nanoparticles were incubated with the corresponding antibodies in a 96-well polypropylene plate for 2 hours, then transferred to a 384-well polystyrene plate and read on a Tecan M1000 spectrophotometer. Absorbance was read from 450 to 650 nm in 2 nm increments, a Microsoft Excel macro was used to determine the absorbance maximum, the data were smoothed, and the data were fit using a quadratic polynomial. The antibody AC-SINS score was determined by subtracting the smoothed maximum absorbance of the average blank (PBS buffer only) from the smoothed maximum absorbance of the antibody sample. A score of less than 8 is considered ideal and indicates a low degree of self-association.

Evaluation of lead CD3 antibody 2B5 variants in diabody-Fc bispecific format

Comprehensive analysis of data from binding assays, AC-SINS and polyreactivity helped narrow down the binding variants to five, which were cloned into bispecific diabodies using anti-tumor sequences and standard cloning strategies well known in the art. Reformatted to Fc molecule. These bispecific antibodies were transiently expressed and purified using standard techniques well known in the art. The bispecific antibodies produced were tested for binding to Jurkat cells, in vitro cytotoxicity (using a T cell retargeting assay), polyreactivity, thermal stability (using DSC) and AC-SINS. Among all five bispecific antibodies, only one bispecific antibody containing the CD3 antibody 2B5 variant 2B5v6 showed strong binding, resulting in strong cytotoxicity. Additionally, these variants showed reduced polyreactivity as assessed by DNA and insulin binding and AC-SINS scores. The score for the 2B5v6 bispecific antibody was less than 8, compared to a value of greater than 8 for the H2B4 bispecific antibody. Based on these results, 2B5v6 was selected as the optimized lead 2B5 variant for further analysis.

Example 7: Clipping evaluation of CDR-H2

Clipping of the VH CDR2 (SEQ ID NO: 16) at position R53 was observed for CD3 antibodies (H2B4 and 2B5) when the bispecific antibodies were expressed in CHO cells but not HEK293 cells. To eliminate the risk from this disadvantage and ensure better uniformity of the product at manufacturing scale, mutations in the clipping region are selected together with other mutations to eliminate the risk from other disadvantages described in the previous examples. Attention was paid to To assess clipping, briefly, diabody-Fc bispecific antibodies were produced using negative control antibody variable domain sequences and CD3 antibody 2B5 or CD3 antibody H2B4 or CD3 antibody 2B5v6. All bispecific diabody-Fc molecules were produced using similar expression and purification processes well known in the art. CHO SSI cell lines were produced using standard procedures. Two chains to encode a bispecific antibody, a dual promoter, and a recombination site for single site integration (SSI) into the CHO cell genome (Zhang, L. et al. Biotechnology Progress. 2015; 31: 1645 -1656]) into the mammalian expression vector pRY19-GA-Q. The produced plasmid was transfected into CHO-K1 SV 10e9 cells through electroporation using the pRY19 vector containing the corresponding gene, and then positive and negative using hygromycin-B and ganciclovir. Selection pressure was performed. These CHO pools underwent a 3-week recovery phase. When observed viability exceeded 90%, a cell bank was produced for future work. The produced CHO pool was then subjected to a 12-day fed-batch platform expression treatment at 1 L scale in CD-CHO medium (ThermoFisher Scientific, Waltham, MA, USA). Day 12 culture harvest was captured on a Protein A column and eluted at low pH. The proA pool was immediately neutralized to pH 8.1. The resulting pure protein was evaluated using capillary gel electrophoresis (cGE) using standard protocols well known in the art. As shown in Table 12 and Figure 7, the 2B5 bispecific antibody showed much reduced clipping compared to H2B4, and while H2B4 showed significantly increased clipping or fragmentation, 2B5v6 showed no clipping. Table 12 shows the results of capillary gel electrophoresis showing fragmentation of the CD3-GUCY2c bispecific antibody. %POI represents peak-of-interest.

[Table 12]

Example 8: GUCY2c-2B5v6 Bispecific Antibody: Expression and Purification of Chimeric CD3-GUCY2c Bispecific Antibody Using CD3 Antibody 2B5v6

Complementary construct pairs (12.5 μg of each chain) were co-transfected into 25 mL log phase cultures containing 1x10 ⁶ cells/mL HEK 293 cells using the ExpiFectamine™ 293 Transfection Kit (Life Technologies). infected. 24 hours after transfection, ExpiFectamine transfection enhancer was added and cells were grown for an additional 4 to 5 days before harvesting. Then, the waste culture was collected and centrifuged to remove cell debris, and then passed through a 20 μm filter.

The clarified conditioned medium containing the GUCY2c T cell bispecific antibody was then purified using protein A affinity chromatography. Samples were loaded onto a 0.45 mL microcolumn (Repligen) pre-packed with MabSelect SuRe Protein A resin (GE Healthcare) using a liquid handler (Tecan). Bound proteins were washed with PBS (pH 7.2), then eluted with 20 mM citric acid, 150 mM sodium chloride (pH 3.5) and neutralized with 2 M tris (pH 8.0). The samples were then desalted into PBS (pH 7.2) using a G25 Sephadex drip column (GE Healthcare) according to the manufacturer's method. Purified proteins were analyzed for purity using analytical size exclusion chromatography using a Mab HTP column (TOSOH) on an Aglient 1200 HPLC according to the manufacturer's protocol. Concentrations were determined by measuring at OD280 nm using a spectrophotometer (Trinean).

Stability evaluation of bispecific antibodies using differential scanning calorimetry

Proteins were diluted to 0.6 mg/mL in phosphate buffered saline (PBS) solution in a volume of 400 μL. PBS was used as a buffer blank in reference cells. PBS contained: 137mM NaCl, 2.7mM KCl, 8.1mM Na2HPO4, and 1.47mM KH2PO4, pH 7.2. Samples were dispensed into the sample tray of the MicroCal VP-Capillary DSC using an autosampler (Malvern Instruments Ltd, Malvern, UK). Samples were equilibrated at 10°C for 5 minutes and then scanned at 100°C/hour up to 110°C. A filtration time of 16 seconds was chosen. Raw data were baseline corrected and protein concentrations were normalized. Data were fit to the MN2-State model with the appropriate number of transitions using Origin software 7.0 (OriginLab Corporation, Northampton, MA, USA). As shown in Figure 8, the GUCY2c-2B5v6 bispecific antibody had a T _m 1 of 70°C, showing excellent thermal stability.

Binding to Naive T Cells

Purified CD3 bispecific antibodies were assayed for cell surface binding to human CD3 on naive human T cells purified from fresh human peripheral blood mononuclear cells (PBMC) using standard flow cytometry methods known in the art using a BD LSRII Fortessa analyzer. It was titrated using . As shown in Figure 9, the GUCY2c-2B5v6 bispecific antibody bound very well to T cells with an EC ₅₀ of binding of 35.44 nM.

Evaluation of bispecific antibody-mediated T cell activity

Human PBMCs were isolated from healthy donor blood using Histopaque-177 (Sigma). Naïve T cells were isolated from PBMC using a T cell enrichment kit (Stem Cell Technologies) (negative selection of T cells). GUCY2c-expressing T84 human tumor cells transfected with luciferase expression constructs were treated with serial dilutions of GUCY2c bispecific antibody or negative control CD3 bispecific antibody along with T cells. The ratio of effector to target cells (E:T) was 10:1 or 5:1. After culturing T cells and tumor cells at 37°C for 48 hours, luciferase signals were measured using neolite reagent (Perkin Elmer). EC ₅₀ values were calculated in Graphpad PRISM using 4-parameter nonlinear regression analysis. Target negative HCT116 or HT29 cells did not show any GUCY2c bispecific antibody mediated cytotoxicity. As shown in Figure 10, GUCY2c-2B5v6 bispecific antibody showed strong cytotoxicity in T cell retargeting assay.

T84 cells were treated with T cells (E:T ratio of 5:1) and GUCY2C-1608 (GUCY2c-2B5v6) at different doses. Supernatants were collected at different time points (24 hours, 48 hours, 96 hours) and analyzed using the multiplex Luminex assay according to the manufacturer's guidelines and read using xPONENT software on a Luminex 200. The cytokines measured were human IFN-gamma, IL10, IL2, IL4, IL6, and TNF-alpha. Figure 11 shows upregulation of (11A)IFN-gamma, (11B)IL10, (11C)IL2, (11D)IL4, (11E)IL6, (11F)TNF-alpha cytokines after activation of T cells by GUCY2c-160. represents.

In vivo evaluation of GUCY2c-2B5v6 bispecific antibody mediated activity

In vivo efficacy studies were performed using an adoptive transfer model. Tumor cell line/patient derived xenograft fragments were transplanted into NSG mice and developed to approximately 200 mm ³ . Mice were administered bispecific compounds intravenously (unless otherwise noted). 2×10 ⁶ activated and expanded T cells (using the T cell expansion and activation kit from Miltenyi Biotec) were administered IV 24 hours after bispecific antibody administration. Bispecific antibodies were administered according to a weekly schedule. As shown in Figures 12 and 13, the GUCY2c-2B5v6 (GUCY2c-1608) bispecific antibody showed excellent long-term tumor regression in PDX CRX-11201 and cell line LS1034 in vivo models indicative of colorectal cancer.

Example 9: FLT3-CD3 bispecific antibody, production of FLT3-2B5v6 bispecific IgG using optimized CD3 antibody (2B5v6), and cytotoxicity

The human FLT3 antibody (xFLT3) and human CD3 antibody (2B5v6) have a antibody that binds to positions 223, 225, and 228 of the hinge region (e.g., (C223E or C223R), (E225E or E225R), and (P228E or P228R)) and human IgG2 antibodies. Expressed individually as human IgG2dA_D265A engineered by EEEE on one arm and RRRR on the other arm for bispecific exchange of positions 409 or 368 of the CH3 region (e.g. K409R or L368E (EU numbering scheme)). Additionally, the antibody had a mutation from D to A at position 265 (EU numbering system).

Individual antibody arms were individually purified using Protein A resin. Then, 1 mg/mL of purified xFLT3 antibody was exchanged for 1 mg/mL of purified 2B5v6 antibody in the presence of 2 mM reduced glutathione. The exchange reaction was carried out at 37°C for 16 hours. Then, 2 mM oxidized glutathione was added to the protein, and the mixture was incubated at 37°C for 30 minutes. The mixture was then diluted 1:5 into 20 mM MES pH 5.4 buffer without NaCl. The diluted sample was loaded onto a MonoS ion exchange column and washed with 20mM MES pH 5.4 25mM NaCl buffer. The bispecific antibody xFLT3-2B5v6 was then eluted by a gradient from 0 to 500 mM NaCl in 20 mM MES pH 5.4 buffer. Finally, the bispecific antibody was dialyzed into phosphate buffered saline (PBS) for cell-based analysis. In cytotoxicity assays, unstimulated human T cells and luciferase expressing target cells (Eol-1 or MV411) were co-cultured at defined ratios in 200 μL of RPMI + 10% FBS in 96 well U-bottom plates. . The xFLT3-2B5v6 bispecific antibody was serially diluted in PBS and added to the wells. After 24 hours, 100 μL of cell suspension was mixed with 100 μL One-GLO reagent in a 96-well white wall plate and luminescence was measured on a photometer. Cytotoxicity values were determined using the formula:

% cytotoxicity = [1- (RLU sample)/RLU target cells only)] x 100RLU

(here RLU: relative light unit).

As shown in Figures 14A and 14B, the xFLT3-2B5v6 bispecific antibody with optimized CD3 antibody (2B5v6) inhibits both cells expressing high copies of FLT3 (EOL-1) and low copies (MV4-11). When tested against it, it appeared very potent in an alternative bispecific IgG format.

Table 13 shows the sequences of the light and heavy chains of FLT3 (EOL-1) and the light and heavy chains of the 2B5v6 bispecific antibody.

[Table 13]

Example 10: T cell epitope risk de-risking of bispecific antibodies

The optimized clone 2B5v6 was analyzed for potential T cell epitopes to eliminate the risk of potential immunogenicity. The Epivax tool for in silico prediction of potential T cell epitopes was used in this analysis. This tool predicts the number of 9-mer frame peptides that bind to any of eight different HLA alleles. A hit is defined as having a Z-score in the top 5%, and a strong hit is defined as having a Z-score in the top 1%. Hits for 4 or more alleles or 1 strong hit are considered as predicted T cell epitopes. Based on these criteria, three predicted non-germline T cell epitopes were identified in CDRH2, two in CDRL1 and one predicted germline epitope in L2 (Table 15). In addition to the scores of individual peptides, the overall risk of a sequence can be defined by the total number of hits or the T-regitope adjusted Epivax score, which has been optimized to correlate with the prevalence of clinical ADA. For these predicted values, the lower the score, the lower the predicted risk of ADA. Here, the VH domain has 70 hits and a score of -42.82, and the VL domain has 60 hits and a score of -23.79.

To reduce the risk of immunogenicity, mutations were identified that could reduce the overall score of these clones, eliminating hits and ideally predicted T cell epitopes. All possible single mutant changes in CDR-L1, CDR-L2, and CDR-H2 were considered to reduce hits, reduce the overall t-regitope adjusted EpiMatrix score, and identify those that could eliminate T cell epitopes. . Because these potential mutations may also affect binding and stability, care was taken to consider the predicted stability and affinity data described above and identify acceptable mutations. Tables 14A, 14B and 14C show mutations designed to reduce the immunogenic risk of 2B5v6. Additionally, the mutations shown in SEQ ID NOs: 44 to 47, 51 to 57, 60 and 62 are predicted to reduce the number of T cell epitopes.

[Table 14A]

[Table 14B]

[Table 14C]

Table 15 shows regions of the CD3 antibody (2B5v6) variable domain that have T cell epitopes and have a potential immunogenic risk.

[Table 15]

All mutants were produced as monovalent IgG antibodies using standard transient HEK293 expression and then purified using Protein A columns. The resulting clones were tested for binding to Jurkat cells expressing CD3 using FACS and for self-association potential using AC-SINS as described in Example 7. As shown in Table 16, five molecules with improved scores, reduced T cell epitopes and maintained affinity for CD3 were selected and tested in the diabody-Fc format. The 2B5v6 variant with improved epivax score was compared with the parent 2B5v6. Results show similar binding and AC-SINS scores for Jurkat cells expressing CD3.

[Table 16]

Among the mutants tested, several mutants showed lower binding to Jurkat cells compared to 2B5v6. Among these mutants, the one with all T cell epitopes removed and the epivax score improved was used to produce a Diabody-Fc bispecific molecule with the GUCY2c antibody as the other binding domain. Table 17 shows the epivax scores and Figure 15 shows reduced binding of these variants to Jurkat cells. The low affinity CD3 antibody 2B5v6 variant shows improved epivax scores.

[Table 17]

Example 11: Three anti-CD3 variants show improved affinity and cytotoxicity in GUCY2C-CD3 bispecific antibody.

Three anti-CD3 variants derived from 2B5v6, 2B5-1038 (SEQ ID NOs: 4 and 9), 2B5-1039 (SEQ ID NOs: 4 and 87), and 2B5-1040 (SEQ ID NOs: 4 and 89), were cloned using standard cloning methods described above. , reformatted into a bispecific diabody-Fc molecule paired with an anti-tumor GUCY2c antibody sequence in one of the arrays shown in Figure 1 using expression and purification techniques. The bispecific antibodies produced shown in Table 18 were tested for in vitro cytotoxicity using a T-cell retargeting assay, binding affinity using surface plasmon resonance (SPR) analysis, and nonspecificity by AC-SINS. did. Immunogenicity risk was assessed by the Epivax tool for in silico prediction of potential T-cell epitopes. Intact mass spectrometry data suggest that all bispecific antibodies are 100% properly paired heterodimers. Table 18 shows that all three bispecific antibodies were (1) approximately 2-fold stronger in in vitro cytotoxicity compared to the control bispecific GUCY2C-1608 paired with anti-CD3 2B5v6 (Figure 16); (2) has a binding affinity for recombinant human CD3 consistent with cytotoxic activity, as measured by SPR; (3) have an improved immunogenicity score compared to the control group; (4) indicates that the AC-SIN score remains the same as the control bispecific GUCY2C-1608. These results show that anti-GUCY2c bispecific antibodies with CD3 variants recruit naïve human T cells and induce apoptosis of T84 tumor cells (Figure 16). The three anti-CD3 variants derived from 2B5v6 showed approximately 2-fold stronger activity compared to the control bispecific GUCY2C-1608.

[Table 18]

The sequences of the bispecific antibodies are shown in Table 19 below.

[Table 19]

ATCC PTA-124943 20180214 ATCC PTA-124944 20180214

SEQUENCE LISTING <110> Pfizer Inc. <120> ANTIBODIES SPECIFIC FOR CD3 AND USES THEREOF <130> PC72375 <140> PCT/IB2019/054186 <141> 2019-05-21 <150> US 62/675,562 <151> 2018-05-23 <150> US 62 /847,460 <151> 2019-05-14 <160> 100 <170> PatentIn version 3.5 <210> 1 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Thr Ser Ser Gln Ser Leu Phe Asn Val 20 25 30 Arg Ser Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Asp Leu Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 2 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 2 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Phe Ile Arg Asn Arg Ala Arg Gly Tyr Thr Ser Asp His Asn Pro 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Asp Arg Pro Ser Tyr Tyr Val Leu Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Val Thr Val Ser Ser 115 120 <210> 3 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 3 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Thr Ser Ser Gln Ser Leu Phe Asn Val 20 25 30 Arg Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Asp Leu Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 4 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 4 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Phe Ile Arg Asn Gln Ala Arg Gly Tyr Thr Ser Asp His Asn Pro 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Asp Arg Pro Ser Tyr Tyr Val Leu Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 5 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 5 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Phe Ile Arg Asn Arg Ala Gln Gly Tyr Thr Ser Asp His Asn Pro 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Asp Arg Pro Ser Tyr Tyr Val Leu Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 6 <211> 112 <212> PRT <213> Artificial Sequence <220> <223 > Synthetic peptide sequence <400> 6 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Thr Ser Ser Gln Ser Leu Phe Asn Val 20 25 30 Arg Ser Gly Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Asp Leu Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 7 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 7 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Phe Ile Arg Asn Gln Ala Arg Gly Tyr Thr Ser Asp His Gln Pro 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Asp Arg Pro Ser Tyr Tyr Val Leu Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Val Thr Val Ser Ser 115 120 <210> 8 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 8 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Thr Ser Ser Gln Ser Leu Phe Asn Val 20 25 30 Arg Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Asp Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Asp Leu Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 9 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 9 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Thr Ser Asp Gln Ser Leu Phe Asn Val 20 25 30 Arg Ser Gly Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Asp Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Asp Leu Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 < 210> 10 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 10 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Phe Ile Arg Asn Gln Asp Arg Gly Tyr Thr Ser Asp His Gln Pro 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Asp Arg Pro Ser Tyr Tyr Val Leu Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 11 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 11 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Thr Ser Ser Gln Ser Leu Phe Asn Val 20 25 30 Arg Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Tyr Leu Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 12 <211> 121 <212 > PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 12 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Phe Ile Arg Asn Gln Ala Arg Gly Tyr Thr Ser Asp His Asn Pro 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Asp Arg His Ser Tyr Tyr Val Leu Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 13 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 13 Asp Tyr Tyr Met Thr 1 5 <210> 14 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 14 Gly Phe Thr Phe Ser Asp Tyr 1 5 <210> 15 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 15 Gly Phe Thr Phe Ser Asp Tyr Tyr Met Thr 1 5 10 <210> 16 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 16 Phe Ile Arg Asn Arg Ala Arg Gly Tyr Thr Ser Asp His Asn Pro Ser 1 5 10 15 Val Lys Gly <210> 17 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 17 Arg Asn Arg Ala Arg Gly Tyr Thr 1 5 <210> 18 <211> 10 <212> PRT <213> Artificial Sequence <220> <223 > Synthetic peptide sequence <400> 18 Asp Arg Pro Ser Tyr Tyr Val Leu Asp Tyr 1 5 10 <210> 19 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 19 Phe Ile Arg Asn Gln Ala Arg Gly Tyr Thr Ser Asp His Asn Pro Ser 1 5 10 15 Val Lys Gly <210> 20 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 20 Arg Asn Gln Ala Arg Gly Tyr Thr 1 5 <210> 21 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 21 Phe Ile Arg Asn Arg Ala Gln Gly Tyr Thr Ser Asp His Asn Pro Ser 1 5 10 15 Val Lys Gly <210> 22 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 22 Arg Asn Arg Ala Gln Gly Tyr Thr 1 5 <210> 23 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 23 Phe Ile Arg Asn Gln Ala Arg Gly Tyr Thr Ser Asp His Gln Pro Ser 1 5 10 15 Val Lys Gly <210> 24 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 24 Phe Ile Arg Asn Gln Asp Arg Gly Tyr Thr Ser Asp His Gln Pro Ser 1 5 10 15 Val Lys Gly <210> 25 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 25 Asp Arg His Ser Tyr Tyr Val Leu Asp Tyr 1 5 10 <210> 26 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 26 Thr Ser Ser Gln Ser Leu Phe Asn Val Arg Ser Arg Lys Asn Tyr Leu 1 5 10 15 Ala <210> 27 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 27 Trp Ala Ser Thr Arg Glu Ser 1 5 <210> 28 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 28 Lys Gln Ser Tyr Asp Leu Phe Thr 1 5 <210> 29 <211> 17 <212> PRT <213 > Artificial Seuqence <220> <223> Synthetic peptide sequence <400> 29 Thr Ser Ser Gln Ser Leu Phe Asn Val Arg Ser Gln Lys Asn Tyr Leu 1 5 10 15 Ala <210> 30 <211> 17 <212> PRT < 213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 30 Thr Ser Ser Gln Ser Leu Phe Asn Val Arg Ser Gly Lys Asn Tyr Leu 1 5 10 15 Ala <210> 31 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 31 Trp Ala Ser Asp Arg Glu Ser 1 5 <210> 32 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 32 Thr Ser Asp Gln Ser Leu Phe Asn Val Arg Ser Gly Lys Asn Tyr Leu 1 5 10 15 Ala <210> 33 <211> 8 <212> PRT <213> Artificial Sequence <220> <223 > Synthetic peptide sequence <400> 33 Lys Gln Ser Tyr Tyr Leu Phe Thr 1 5 <210> 34 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 34 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Thr Ser Ser Gln Ser Leu Phe Asn Val 20 25 30 Arg Ser Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Asp Leu Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 35 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 35 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Phe Ile Arg Asn Gln Ala Arg Gly Tyr Thr Ser Asp His Asn Pro 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Asp Arg Pro Ser Tyr Tyr Val Leu Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 36 <211> 461 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 36 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Tyr Tyr 20 25 30 Gly Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Asn Val Glu Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Arg 85 90 95 Lys Val Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly 100 105 110 Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly 115 120 125 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 130 135 140 Phe Thr Phe Ser Asp Tyr Tyr Met Thr Trp Val Arg Gln Ala Pro Gly 145 150 155 160 Lys Gly Leu Glu Trp Val Ala Phe Ile Arg Asn Arg Ala Arg Gly Tyr 165 170 175 Thr Ser Asp His Asn Pro Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 180 185 190 Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala 195 200 205 Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg Pro Ser Tyr Tyr 210 215 220 Val Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly 225 230 235 240 Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe 245 250 255 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 260 265 270 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 275 280 285 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 290 295 300 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 305 310 315 320 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 325 330 335 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 340 345 350 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro 355 360 365 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val 370 375 380 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 385 390 395 400 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 405 410 415 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 420 425 430 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 435 440 445 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 450 455 460 <210> 37 <211> 464 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence < 400> 37 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Phe Asn Val 20 25 30 Arg Ser Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Ser Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Asp Leu Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 Gly Gly Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly 115 120 125 Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala 130 135 140 Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Met His Trp Val Arg Gln Ala 145 150 155 160 Pro Gly Lys Gly Leu Glu Trp Ile Gly Glu Ile Lys Pro Ser Asn Gly 165 170 175 Leu Thr Asn Tyr Ile Glu Lys Phe Lys Asn Arg Phe Thr Ile Ser Val 180 185 190 Asp Lys Ala Lys Asn Ser Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala 195 200 205 Glu Asp Thr Ala Val Tyr Tyr Cys Thr Arg Thr Ile Thr Thr Glu 210 215 220 Gly Tyr Trp Phe Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val 225 230 235 240 Ser Ser Gly Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro 245 250 255 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 260 265 270 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 275 280 285 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 290 295 300 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 305 310 315 320 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 325 330 335 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 340 345 350 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 355 360 365 Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser 370 375 380 Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 385 390 395 400 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 405 410 415 Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys 420 425 430 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 435 440 445 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 450 455 460 <210> 38 <211> 464 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 38 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Thr Ser Ser Gln Ser Leu Phe Asn Val 20 25 30 Arg Ser Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Asp Leu Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Gly Gly Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly 115 120 125 Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala 130 135 140 Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Met His Trp Val Arg Gln Ala 145 150 155 160 Pro Gly Lys Gly Leu Glu Trp Ile Gly Glu Ile Lys Pro Ser Asn Gly 165 170 175 Leu Thr Asn Tyr Ile Glu Lys Phe Lys Asn Arg Phe Thr Ile Ser Val 180 185 190 Asp Lys Ala Lys Asn Ser Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala 195 200 205 Glu Asp Thr Ala Val Tyr Tyr Cys Thr Arg Thr Ile Thr Thr Thr Glu 210 215 220 Gly Tyr Trp Phe Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val 225 230 235 240 Ser Ser Gly Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro 245 250 255 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 260 265 270 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 275 280 285 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 290 295 300 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 305 310 315 320 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 325 330 335 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 340 345 350 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 355 360 365 Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser 370 375 380 Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 385 390 395 400 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 405 410 415 Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys 420 425 430 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 435 440 445 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 450 455 460 <210> 39 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 39 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 40 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 40 Met Gln Ser Gly Thr His Trp Arg Val Leu Gly Leu Cys Leu Leu Ser 1 5 10 15 Val Gly Val Trp Gly Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr 20 25 30 Gln Thr Pro Tyr Lys Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr 35 40 45 Cys Pro Gln Tyr Pro Gly Ser Glu Ile Leu Trp Gln His Asn Asp Lys 50 55 60 Asn Ile Gly Gly Asp Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp 65 70 75 80 His Leu Ser Leu Lys Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr 85 90 95 Val Cys Tyr Pro Arg Gly Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu 100 105 110 Tyr Leu Arg Ala Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Pro Ile Glu Glu Leu Glu Asp Arg Val Phe Val Asn 130 135 140 Cys Asn Thr Ser Ile Thr Trp Val Glu Gly Thr Val Gly Thr Leu Leu 145 150 155 160 Ser Asp Ile Thr Arg Leu Asp Leu Gly Lys Arg Ile Leu Asp Pro Arg 165 170 175 Gly Ile Tyr Arg Cys Asn Gly Thr Asp Ile Tyr Lys Asp Lys Glu Ser 180 185 190 Thr Val Gln Val His Tyr Arg Met Cys Gln Ser Cys Val Glu Leu Asp 195 200 205 His His His His His His 210 <210> 41 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 41 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Thr Phe Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 42 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 42 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Gly Gly Arg Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Leu Ser Pro Ser Asp Val Gly Trp Gly Tyr Gly Phe Asp 100 105 110 Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 43 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 43 Ala Ala Ser Ser Leu Gln Ser 1 5 <210> 44 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 44 Val Gln Pro Gly Gly Ser Leu Arg Leu 1 5 <210> 45 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 45 Leu Arg Leu Ser Cys Ala Ala Ser Gly 1 5 <210> 46 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 46 Trp Val Arg Gln Ala Pro Gly Lys Gly 1 5 <210> 47 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 47 Val Arg Gln Ala Pro Gly Lys Gly Leu 1 5 <210> 48 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 48 Trp Val Ala Phe Ile Arg Asn Gln Ala 1 5 <210> 49 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 49 Phe Ile Arg Asn Gln Ala Arg Gly Tyr 1 5 <210> 50 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 50 Tyr Thr Ser Asp His Asn Pro Ser Val 1 5 <210> 51 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 51 Val Lys Gly Arg Phe Thr Ile Ser Arg 1 5 <210> 52 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 52 Phe Thr Ile Ser Arg Asp Asn Ala Lys 1 5 <210> 53 <211> 9 <212> PRT <213> Artificial Sequence <220> < 223> Synthetic peptide sequence <400> 53 Leu Tyr Leu Gln Met Asn Ser Leu Arg 1 5 <210> 54 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 54 Tyr Leu Gln Met Asn Ser Leu Arg Ala 1 5 <210> 55 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 55 Leu Gln Met Asn Ser Leu Arg Ala Glu 1 5 <210> 56 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 56 Ile Gln Met Thr Gln Ser Pro Ser Ser 1 5 <210> 57 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 57 Met Thr Gln Ser Pro Ser Ser Leu Ser 1 5 <210> 58 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 58 Ile Thr Cys Thr Ser Ser Gln Ser Leu 1 5 <210> 59 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 59 Val Arg Ser Gln Lys Asn Tyr Leu Ala 1 5 <210> 60 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 60 Trp Tyr Gln Gln Lys Pro Gly Lys Ala 1 5 <210> 61 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 61 Leu Leu Ile Tyr Trp Ala Ser Thr Arg 1 5 <210 > 62 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 62 Phe Thr Leu Thr Ile Ser Ser Leu Gln 1 5 <210> 63 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 63 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15 <210> 64 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 64 Cys Pro Pro Cys Pro 1 5 <210> 65 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 65 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 <210> 66 <211> 186 <212> PRT <213> Homo sapiens <400> 66 Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr Gln Thr Pro Tyr Lys 1 5 10 15 Val Ser Ile Ser Gly Thr Val Ile Leu Thr Cys Pro Gln Tyr Pro 20 25 30 Gly Ser Glu Ile Leu Trp Gln His Asn Asp Lys Asn Ile Gly Gly Asp 35 40 45 Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp His Leu Ser Leu Lys 50 55 60 Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr Val Cys Tyr Pro Arg 65 70 75 80 Gly Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu Tyr Leu Arg Ala Arg 85 90 95 Val Cys Glu Asn Cys Met Glu Met Asp Val Met Ser Val Ala Thr Ile 100 105 110 Val Ile Val Asp Ile Cys Ile Thr Gly Gly Leu Leu Leu Leu Val Tyr 115 120 125 Tyr Trp Ser Lys Asn Arg Lys Ala Lys Ala Lys Pro Val Thr Arg Gly 130 135 140 Ala Gly Ala Gly Gly Arg Gln Arg Gly Gln Asn Lys Glu Arg Pro Pro 145 150 155 160 Pro Val Pro Asn Pro Asp Tyr Glu Pro Ile Arg Lys Gly Gln Arg Asp 165 170 175 Leu Tyr Ser Gly Leu Asn Gln Arg Arg Ile 180 185 <210> 67 <211> 1534 <212> DNA <213> Homo sapiens <400> 67 tattgtcaga gtcctcttgt ttggccttct aggaaggctg tgggaccca g ctttcttcaa 60 ccagtccagg tggaggcctc tgccttgaac gtttccaagt gaggtaaaac ccgcaggccc 120 agaggcctct ctacttcctg tgtggggttc agaaaccctc ctcccctccc agcctcaggt 180 gcctgcttca gaaaatgaag tagtaagtct gctggcctcc gccatcttag taaagtaaca 240 gtcccatgaa a caaagatgc agtcgggcac tcactggaga gttctgggcc tctgcctctt 300 atcagttggc gtttgggggc aagatggtaa tgaagaaatg ggtggtatta cacagacacc 360 atataaagtc tccatctctg gaaccacagt aatattgaca tgccctcagt atcctggatc 420 tgaaat acta tggcaacaca atgataaaaa cataggcggt gatgaggatg ataaaaaacat 480 aggcagtgat gaggatcacc tgtcactgaa ggaattttca gaattggagc aaagtggtta 540 ttatgtctgc taccccagag gaagcaaacc agaagatgcg aacttttatc tctacctgag 600 ggcaagagtg tgtgagaact gcatggagat ggatgtgatg tcggtggcca caattgtcat 660 agtggacatc tgcatcactg gggg cttgct gctgctggtt tactactgga gcaagaatag 720 aaaggccaag gccaagcctg tgacacgagg agcgggtgct ggcggcaggc aaaggggaca 780 aaacaaaggag aggccaccac ctgttcccaa cccagactat gagcccatcc ggaaaggcca 840 gcgggacctg tattctggcc tgaatca gag acgcatctga ccctctggag aacactgcct 900 cccgctggcc caggtctcct ctccagtccc cctgcgactc cctgtttcct gggctagtct 960 tggaccccac gagagagaat cgttcctcag cctcatggtg aactcgcgcc ctccagcctg 1020 atcccccgct ccctcctccc tgccttctct gctggtaccc agtcctaaaa tattgctgct 1080 tcctcttcct ttgaagcatc atcagtag tc acaccctcac agctggcctg ccctcttgcc 1140 aggatattta tttgtgctat tcactccctt ccctttggat gtaacttctc cgttcagttc 1200 cctccttttc ttgcatgtaa gttgtccccc atcccaaagt attccatcta cttttctatc 1260 gcc gtcccct tttgcagccc tctctgggga tggactgggt aaatgttgac agaggccctg 1320 ccccgttcac agatcctggc cctgagccag ccctgtgctc ctccctcccc caacactccc 1380 taccaacccc ctaatcccct actccctcca ccccccctcc actgtaggcc actggatggt 1440 catttgcatc tccgtaaatg tgctctgctc ctcagctgag agagaaaaaa ataaactgta 1500 tttggctgca agaaaaaaaaa aaaaaaaaaa aaaa 1534 <210> 68 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleotide sequence <400> 68 gacatccaga tgacccagtc cccctcttct ctgtctgcct ctgtgggcga cagagtgacc 60 atcacctgca caagctcaca gtcactgttt aatgtccgca gccggaaaaaa ctatcttgcg 120 tggtatcagc agaagcctgg caaggctccc aagctgctga tct actgggc cagtacacga 180 gaatccggcg tgccttccag attctccggc tctggctctg gcaccgattt caccctgacc 240 atctcctccc tccagcctga ggatttcgcc acctactact gcaaacagtc ttacgacctt 300 ttcacttttg gcggcggaac aaaggtggag atcaag 3 36 <210> 69 <211 > 363 <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleotide sequence <400> 69 gaagtgcagc ttgttgaatc tggcggcggt ttggttcagc ccggtggatc actgcgactc 60 agttgcgcag ctagcggctt caccttttct gattactaca tga catgggt acgacaggcg 120 ccaggcaagg gtttggaatg ggtagcattc atacgcaata gggcacgcgg gtacacttca 180 gaccacaatc cctcagtaaa aggaagattt accatctcaa gagacaatgc caaaaattca 240 ctctacctgc aaatgaactc acttcgcgcc gaggataccg ccgtgtatta ctgtgccaga 300 gacagaccat cttattacgt gctggactat tggggacagg gcactacagt caccgtcagc 360 tct 363 <210> 70 <211> 336 <212> DNA <2 13> Artificial Sequence <220> <223> Synthetic nucleotide sequence <400> 70 gacatccaga tgacccagtc cccctcttct ctgtctgcct ctgtgggcga cagagtgacc 60 atcacctgca caagctcaca gtcactgttt aatgtccgca gccagaaaaa ctatcttgcg 120 tggtatcagc agaagcctgg caaggctccc aagctgctga tctactgggc cagtacacga 180 gaatccggcg tgccttccag attctccggc tctggctctg gcaccgattt caccctgacc 240 atctcctccc tccagcctga ggatttcgcc acctactact gcaaacagtc ttacgacctt 300 ttcacttttg gcggcggaac aaaggtggag atcaag 336 <210> 71 <211> 363 <2 12> DNA <213> Artificial Sequence <220> <223> Synthetic nucleotide sequence <400> 71 gaagtgcagc ttgttgaatc tggcggcggt ttggttcagc ccggtggatc actgcgactc 60 agttgcgcag ctagcggctt caccttttct gattactaca tgacatgggt acgacaggcg 120 ccaggcaagg gtttggaatg ggtagcattc atacgcaatc aggcacgcgg gtacacttca 180 gaccacaatc cctcagtaaa aggaagattt accatctcaa gagacaatgc caaaaattca 240 ctctacctgc aaatgaactc acttcgcgcc gaggataccg ccgtgtatta ctgtgccaga 300 gacagaccat cttattacgt gctggactat tggggacagg gcactacagt caccgtcagc 360 tct 363 <210> 72 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleotide sequence <400> 72 gaagtgcagc ttgttgaatc tggcggcggt ttggttcagc ccggtggatc actgcgactc 60 agttgcgcag ctagcggctt caccttttct gattactaca tgacatgggt acgacaggcg 120 ccaggcaagg gtttggaatg ggtagcattc atacgcaata gggcacaggg gtacacttca 180 gaccacaatc cctcagtaaa aggaagattt accatctcaa gagacaatgc caaaaattca 240 ctctacctgc a aatgaactc acttcgcgcc gaggataccg ccgtgtatta ctgtgccaga 300 gacagaccat cttattacgt gctggactat tggggacagg gcactacagt caccgtcagc 360 tct 363 <210> 73 <211> 363 <212> DNA <213 > Artificial Sequence <220> <223> Synthetic nucleotide sequence <400> 73 gaagtgcagc ttgttgaatc tggcggcggt ttggttcagc ccggtggatc actgcgactc 60 agttgcgcag ctagcggctt caccttttct gattactaca tgacatgggt acgacaggcg 120 ccaggcaagg gtttggaatg ggtagcattc atacgcaatc aggcacgcgg gtacacttca 180 gaccaccagc cctcagtaaa aggaagattt accatctcaa gagacaatgc caaaaattca 240 ctctacctgc aaatgaactc acttcgcgcc gaggataccg ccgtgtatta ctgtgccaga 300 gacagaccat cttattacgt gctggactat tggggacagg gcactacagt caccgtcagc 360 tct 363 <210> 74 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleotide sequence <400> 74 gacatccaga tgacccagtc cccct cttct ctgtctgcct ctgtgggcga cagagtgacc 60 atcacctgca caagctcaca gtcactgttt aatgtccgca gccagaaaaa ctatcttgcg 120 tggtatcagc agaagcctgg caaggctccc aagctgctga tctactgggc cagtgatcga 180 gaatccggcg tgccttccag attctccggc tctggctctg gcaccgattt caccctgacc 240 atctcctccc tccagcctga ggatt tcgcc acctactact gcaaacagtc ttacgacctt 300 ttcacttttg gcggcggaac aaaggtggag atcaag 336 <210> 75 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleotide sequence <400> 75 gacatccaga tgacccagtc cccctcttct ctgtctgcct ctgtgggcga cagagtgacc 60 atcacctgca caagcgacca gtcactgttt aatgtccgca gcggcaaaaa ctatcttgcg 120 tggtatcagc agaagcctgg caagg ctccc aagctgctga tctactgggc cagtgaccga 180 gaatccggcg tgccttccag attctccggc tctggctctg gcaccgattt caccctgacc 240 atctcctccc tccagcctga ggatttcgcc acctactact gcaaacagtc ttacgacctt 300 ttcacttttg gcggcggaac aaaggtggag atcaag 336 <210> 76 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleotide sequence <400> 76 gaagtgcagc ttgttgaatc tggcggcggt ttggttcagc ccggtggatc actgcgactc 60 agttgcgcag ctagcggctt cacc ttttct gattactaca tgacatgggt acgacaggcg 120 ccaggcaagg gtttggaatg ggtagcattc atacgcaatc aggaccgcgg gtacacttca 180 gaccaccagc cctcagtaaa aggaagattt accatctcaa gagacaatgc caaaaattca 240 ctctacctgc aaatgaactc acttcgcgcc gaggataccg ccgtgtatta ctgtgccaga 300 gacagaccat cttattacgt gctggactat tggggacagg gcactacagt caccgtcagc 3 60 tct 363 <210> 77 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleotide sequence <400> 77 gacatccaga tgacccagtc cccctcttct ctgtctgcct ctgtgggcga cagagtgacc 60 atcacctgca caagctcaca gtcactgttt aatgtccgca gccagaaaaa ctatcttgcg 120 tggtatcagc agaagcctgg caaggctccc aagctgctga tctact gggc cagtacacga 180 gaatccggcg tgccttccag attctccggc tctggctctg gcaccgattt caccctgacc 240 atctcctccc tccagcctga ggatttcgcc acctactact gcaaacagtc ttactacctt 300 ttcacttttg gcggcggaac aaaggtggag atcaag 336 <210> 78 <211 > 216 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 78 Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Glu Glu Met 115 120 125 Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly 210 215 <210> 79 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 79 Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met 115 120 125 Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly 210 215 <210> 80 <211> 326 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 80 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Arg Val Arg Cys Pro Arg Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Ala 130 135 140 Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205 Ser Ser Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly Lys 325 <210> 81 <211> 326 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 81 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Glu Val Glu Cys Pro Glu Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Ala 130 135 140 Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205 Ser Ser Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Glu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly Lys 325 <210> 82 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 82 Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 115 120 125 Thr Lys Asn Gln Val Ser Leu Ser Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly 210 215 <210> 83 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleotide sequence <400> 83 gacatccaga tgacccagtc cccctcttct ctgtctgcct ctgtgggcga cagagtgacc 60 atcacct gca caagctcaca gtcactgttt aatgtccgca gcggaaaaaa ctatcttgcg 120 tggtatcagc agaagcctgg caaggctccc aagctgctga tctactgggc cagtacacga 180 gaatccggcg tgccttccag attctccggc tctggctctg gcaccgattt caccctgacc 240 atctcctccc tccagcctga ggatttcgcc acctactact gcaaac agtc ttacgacctt 300 ttcacttttg gcggcggaac aaaggtggag atcaag 336 <210> 84 <211> 363 <212> DNA <213> Artificial Sequence <220> <223 > Synthetic nucleotide sequence <400> 84 gaagtgcagc ttgttgaatc tggcggcggt ttggttcagc ccggtggatc actgcgactc 60 agttgcgcag ctagcggctt caccttttct gattactaca tgacatgggt acgacaggcg 120 ccaggcaagg gtttggaatg gg tagcattc atacgcaatc aggcacgcgg gtacacttca 180 gaccacaatc cctcagtaaa aggaagattt accatctcaa gagacaatgc caaaaattca 240 ctctacctgc aaatgaactc acttcgcgcc gaggataccg ccgtgtatta ctgtgccaga 300 gacagacact cttattacgt gctggact at tggggacagg gcactacagt caccgtcagc 360 tct 363 <210> 85 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 85 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Phe Asn Val 20 25 30 Arg Ser Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Ser Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Asp Leu Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 86 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleotide sequence <400> 86 gacatcgtga tgacccagag ccccgatagc ctggccgtgt ctctgggaga gagagccacc 60 atcaactgca agagcagcca gagcctgttc aacgtgagaa gccggaagaa ctacctggcc 120 tggtatcagc agaaacccgg ccagcccccc aagctgctga tcagctgggc cagcaccaga 180 gaaagcggcg tgcccgatag attca gcggc agcggaagcg gcaccgactt caccctgaca 240 atcagctccc tgcaggccga ggacgtggcc gtgtactact gcaagcagag ctacgacctg 300 ttcaccttcg gcagcggcac caagctggaa atcaaa 336 <210> 87 <211> 112 <212> PRT < 213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 87 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Thr Ser Ser Glu Ser Leu Phe Asn Val 20 25 30 Arg Ser Gly Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Asp Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Asp Leu Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 88 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleotide sequence <400> 88 gacatccaga tgacccagtc cccctcttct ctgtctgcct ctgtgggcga cagagtgacc 60 atcacctgca caagctcaga gtcactgttt aatgtccgca gcggcaaaaa ctatcttgcg 120 tggtatcagc agaagcctgg caaggctccc aagctgctga tctactgggc cagtgaccga 180 gaatccggcg tgccttccag attctccggc tctggctctg gcaccgattt caccctgacc 240 atctcctccc tccagcctga ggatttcgcc acctactact gcaaacagtc ttacgacctt 300 ttcacttttg gcggcggaac aaaggtggag atcaag 336 <210> 89 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 89 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Thr Ser Ser Gln Ser Leu Phe Asn Val 20 25 30 Arg Ser Gly Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Asp Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Asp Leu Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 90 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleotide sequence < 400> 90 gacatccaga tgacccagtc cccctcttct ctgtctgcct ctgtgggcga cagagtgacc 60 atcacctgca caagctcaca gtcactgttt aatgtccgca gcggcaaaaa ctatcttgcg 120 tggtatcagc agaagcctgg caaggctccc aagctgctga tct actgggc cagtgaccga 180 gaatccggcg tgccttccag attctccggc tctggctctg gcaccgattt caccctgacc 240 atctcctccc tccagcctga ggatttcgcc acctactact gcaaacagtc ttacgacctt 300 ttcacttttg gcggcggaac aaaggtggag atcaag 3 36 <210> 91 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 91 Thr Ser Ser Glu Ser Leu Phe Asn Val Arg Ser Gly Lys Asn Tyr Leu 1 5 10 15 Ala <210> 92 <211 > 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 92 Thr Ser Ser Gln Ser Leu Phe Asn Val Arg Ser Gly Lys Asn Tyr Leu 1 5 10 15 Ala <210> 93 < 211> 461 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 93 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Tyr Tyr 20 25 30 Gly Ser Ser Leu Leu Gln Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Lys Leu Ala Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Arg 85 90 95 Lys Ala Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly 100 105 110 Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly 115 120 125 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 130 135 140 Phe Thr Phe Ser Asp Tyr Tyr Met Thr Trp Val Arg Gln Ala Pro Gly 145 150 155 160 Lys Gly Leu Glu Trp Val Ala Phe Ile Arg Asn Gln Ala Arg Gly Tyr 165 170 175 Thr Ser Asp His Asn Pro Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 180 185 190 Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala 195 200 205 Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg Pro Ser Tyr Tyr 210 215 220 Val Leu Asp Tyr Trp Gly Gln Gly Thr Val Thr Val Ser Ser Gly 225 230 235 240 Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe 245 250 255 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 260 265 270 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 275 280 285 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 290 295 300 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 305 310 315 320 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 325 330 335 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 340 345 350 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro 355 360 365 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val 370 375 380 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 385 390 395 400 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 405 410 415 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 420 425 430 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 435 440 445 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 450 455 460 <210> 94 <211> 465 <212> PRT <213> Artificial Sequence <220> < 223> Synthetic peptide sequence <400> 94 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Thr Ser Ser Gln Ser Leu Phe Asn Val 20 25 30 Arg Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Asp Leu Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser 115 120 125 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 130 135 140 Ala Ser Gly Phe Thr Phe Ser Ser Tyr Trp Met His Trp Val Arg Gln 145 150 155 160 Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Glu Ile Lys Pro Ser Asn 165 170 175 Glu Leu Thr Asn Val His Glu Lys Phe Lys Asp Arg Phe Thr Ile Ser 180 185 190 Val Asp Lys Ala Lys Asn Ser Ala Tyr Leu Gln Met Asn Ser Leu Arg 195 200 205 Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr Arg Thr Ile Thr Thr Thr 210 215 220 Glu Gly Tyr Trp Phe Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Gly Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala 245 250 255 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 260 265 270 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 275 280 285 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 290 295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 340 345 350 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 355 360 365 Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 370 375 380 Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 405 410 415 Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp 420 425 430 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 435 440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 450 455 460 Gly 465 <210> 95 <211> 461 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 95 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Tyr Tyr 20 25 30 Gly Ser Ser Leu Leu Gln Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Lys Leu Ala Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Arg 85 90 95 Lys Ala Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly 100 105 110 Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly 115 120 125 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 130 135 140 Phe Thr Phe Ser Asp Tyr Tyr Met Thr Trp Val Arg Gln Ala Pro Gly 145 150 155 160 Lys Gly Leu Glu Trp Val Ala Phe Ile Arg Asn Gln Ala Arg Gly Tyr 165 170 175 Thr Ser Asp His Asn Pro Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 180 185 190 Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala 195 200 205 Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg Pro Ser Tyr Tyr 210 215 220 Val Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly 225 230 235 240 Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe 245 250 255 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 260 265 270 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 275 280 285 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 290 295 300 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 305 310 315 320 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 325 330 335 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 340 345 350 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro 355 360 365 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val 370 375 380 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 385 390 395 400 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 405 410 415 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 420 425 430 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 435 440 445 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 450 455 460 <210> 96 <211> 465 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 96 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Thr Ser Asp Gln Ser Leu Phe Asn Val 20 25 30 Arg Ser Gly Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Asp Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Asp Leu Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser 115 120 125 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 130 135 140 Ala Ser Gly Phe Thr Phe Ser Ser Tyr Trp Met His Trp Val Arg Gln 145 150 155 160 Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Glu Ile Lys Pro Ser Asn 165 170 175 Glu Leu Thr Asn Val His Glu Lys Phe Lys Asp Arg Phe Thr Ile Ser 180 185 190 Val Asp Lys Ala Lys Asn Ser Ala Tyr Leu Gln Met Asn Ser Leu Arg 195 200 205 Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr Arg Thr Ile Thr Thr 210 215 220 Glu Gly Tyr Trp Phe Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Gly Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala 245 250 255 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 260 265 270 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 275 280 285 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 290 295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 340 345 350 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 355 360 365 Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 370 375 380 Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu Ser Asn Gly Gln Pro Glu Asn Tyr Lys Thr Thr Pro Pro Val 405 410 415 Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp 420 425 430 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 435 440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 450 455 460 Gly 465 <210> 97 <211> 461 < 212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 97 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Tyr Tyr 20 25 30 Gly Ser Ser Leu Leu Gln Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Lys Leu Ala Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Arg 85 90 95 Lys Ala Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly 100 105 110 Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly 115 120 125 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 130 135 140 Phe Thr Phe Ser Asp Tyr Tyr Met Thr Trp Val Arg Gln Ala Pro Gly 145 150 155 160 Lys Gly Leu Glu Trp Val Ala Phe Ile Arg Asn Gln Ala Arg Gly Tyr 165 170 175 Thr Ser Asp His Asn Pro Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 180 185 190 Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala 195 200 205 Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg Pro Ser Tyr Tyr 210 215 220 Val Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly 225 230 235 240 Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe 245 250 255 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 260 265 270 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 275 280 285 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 290 295 300 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 305 310 315 320 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 325 330 335 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 340 345 350 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro 355 360 365 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val 370 375 380 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 385 390 395 400 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 405 410 415 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 420 425 430 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 435 440 445 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 450 455 460 <210> 98 <211> 465 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 98 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Thr Ser Ser Gln Ser Leu Phe Asn Val 20 25 30 Arg Ser Gly Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Asp Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Asp Leu Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser 115 120 125 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 130 135 140 Ala Ser Gly Phe Thr Phe Ser Ser Tyr Trp Met His Trp Val Arg Gln 145 150 155 160 Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Glu Ile Lys Pro Ser Asn 165 170 175 Glu Leu Thr Asn Val His Glu Lys Phe Lys Asp Arg Phe Thr Ile Ser 180 185 190 Val Asp Lys Ala Lys Asn Ser Ala Tyr Leu Gln Met Asn Ser Leu Arg 195 200 205 Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr Arg Thr Ile Thr Thr Thr 210 215 220 Glu Gly Tyr Trp Phe Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Gly Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala 245 250 255 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 260 265 270 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 275 280 285 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 290 295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 340 345 350 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 355 360 365 Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 370 375 380 Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Pro Val 405 410 415 Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp 420 425 430 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 435 440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 450 455 460 Gly 465 <210> 99 <211> 461 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 99 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Tyr Tyr 20 25 30 Gly Ser Ser Leu Leu Gln Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Lys Leu Ala Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Arg 85 90 95 Lys Ala Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly 100 105 110 Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly 115 120 125 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 130 135 140 Phe Thr Phe Ser Asp Tyr Tyr Met Thr Trp Val Arg Gln Ala Pro Gly 145 150 155 160 Lys Gly Leu Glu Trp Val Ala Phe Ile Arg Asn Gln Ala Arg Gly Tyr 165 170 175 Thr Ser Asp His Asn Pro Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 180 185 190 Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala 195 200 205 Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg Pro Ser Tyr Tyr 210 215 220 Val Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly 225 230 235 240 Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe 245 250 255 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 260 265 270 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 275 280 285 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 290 295 300 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 305 310 315 320 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 325 330 335 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 340 345 350 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro 355 360 365 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val 370 375 380 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 385 390 395 400 Gln Pro Glu Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 405 410 415 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 420 425 430 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 435 440 445 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 450 455 460 <210> 100 <211> 465 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 100 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Thr Ser Ser Glu Ser Leu Phe Asn Val 20 25 30 Arg Ser Gly Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Asp Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Cys Lys Gln 85 90 95 Ser Tyr Asp Leu Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser 115 120 125 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 130 135 140 Ala Ser Gly Phe Thr Phe Ser Ser Tyr Trp Met His Trp Val Arg Gln 145 150 155 160 Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Glu Ile Lys Pro Ser Asn 165 170 175 Glu Leu Thr Asn Val His Glu Lys Phe Lys Asp Arg Phe Thr Ile Ser 180 185 190 Val Asp Lys Ala Lys Asn Ser Ala Tyr Leu Gln Met Asn Ser Leu Arg 195 200 205 Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr Arg Thr Ile Thr Thr Thr 210 215 220 Glu Gly Tyr Trp Phe Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Gly Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala 245 250 255 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 260 265 270 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 275 280 285 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 290 295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 340 345 350 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 355 360 365 Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 370 375 380 Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Pro Val 405 410 415 Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp 420 425 430 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 435 440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 450 455 460Gly 465

Claims (30)

a. (i) heavy chain variable (VH) complementation determining region 1 (VH CDR1) comprising the sequence of SEQ ID NO: 13, 14 or 15; (ii) VH complementation determining region 2 (VH CDR2) comprising the sequence of SEQ ID NO: 19 or 20; and (iii) a VH region comprising VH complementation determining region 3 (VH CDR3) comprising the sequence of SEQ ID NO: 18; and
b. (i) light chain variable (VL) complementation determining region 1 (VL CDR1) comprising the sequence of SEQ ID NO: 29; (ii) VL complementation determining region 2 (VL CDR2) comprising the sequence of SEQ ID NO: 27; and (iii) a VL region comprising VL complementation determining region 3 (VL CDR3) comprising the sequence of SEQ ID NO: 28.
An antibody that specifically binds to CD3, including. According to paragraph 1,
a. The VH region comprises the sequence of SEQ ID NO:4;
b. An antibody, wherein the VL region comprises the sequence of SEQ ID NO:3. According to paragraph 1,
Fab, Fab fragment, F(ab) ₂ fragment, Fv fragment, single chain Fv fragment, disulfide stabilized Fv fragment, single chain antibody, monoclonal antibody, chimeric antibody, bispecific antibody, trispecific antibody, multispecific An antibody selected from the group consisting of antibodies, bispecific heterodimeric diabodies, bispecific heterodimeric IgGs, polyclonal antibodies, labeled antibodies, humanized antibodies, human antibodies and fragments thereof. According to paragraph 3,
An antibody that is a bispecific antibody. In number 4,
A bispecific antibody that specifically binds to a tumor antigen. According to paragraph 1,
An antibody further comprising a human VH framework or a humanized VH framework, and a human VL framework or a humanized VL framework. According to paragraph 1,
Antibodies, which are humanized antibodies. A pharmaceutical composition for treating cancer comprising the antibody of any one of claims 1 to 3, 6, and 7 and a pharmaceutically acceptable carrier. According to clause 8,
A pharmaceutical composition, wherein the cancer is selected from the group consisting of breast cancer, ovarian cancer, thyroid cancer, prostate cancer, cervical cancer, lung cancer, bladder cancer, endometrial cancer, head and neck cancer, testicular cancer, glioblastoma, and cancer of the digestive system. According to clause 9,
A pharmaceutical composition, wherein the cancer of the digestive system is selected from the group consisting of esophageal cancer, stomach cancer, small intestine cancer, colon cancer, rectal cancer, anal cancer, liver cancer, gallbladder cancer, appendix cancer, bile duct cancer, and pancreatic cancer. According to any one of claims 1 to 3, 6 and 7,
An antibody, for use in a treatment involving activation of a cytolytic T cell response. According to clause 8,
A pharmaceutical composition for use in a treatment involving activation of a cytolytic T cell response. According to any one of claims 1 to 3, 6 and 7,
An antibody for the manufacture of a medicament for use in therapy involving activation of a cytolytic T cell response. According to clause 8,
A pharmaceutical composition for the manufacture of a medicament for use in a treatment involving activation of a cytolytic T cell response. A polynucleotide comprising a nucleotide sequence encoding the antibody of any one of claims 1 to 3, 6, and 7. A vector containing the polynucleotide of claim 15. A host cell containing the vector of claim 16. According to clause 17,
A host cell that recombinantly produces the antibody. According to clause 17,
A host cell selected from the group consisting of bacterial cell lines, mammalian cell lines, insect cell lines, and yeast cell lines. According to clause 17,
The host cell is a mammalian cell line, and the mammalian cell line is a CHO cell line. A method for producing an antibody, comprising culturing the host cell of claim 17 and purifying the antibody from the culture supernatant. According to any one of claims 1 to 3, 6 and 7,
An antibody, for use in a method of inhibiting the growth of tumor cells in a subject. According to clause 8,
A pharmaceutical composition for use in a method of inhibiting the growth of tumor cells in a subject. According to any one of claims 1 to 3, 6 and 7,
An antibody for use in the treatment of cancer, optionally wherein said cancer is the group consisting of breast cancer, ovarian cancer, thyroid cancer, prostate cancer, cervical cancer, lung cancer, bladder cancer, endometrial cancer, head and neck cancer, testicular cancer, glioblastoma, and cancer of the digestive system. An antibody selected from: According to any one of claims 1 to 3, 6 and 7,
An antibody for use in the treatment of cancer, wherein a T cell mediated immune response is modulated or the growth of tumor cells is inhibited. According to clause 8,
A pharmaceutical composition for use in the treatment of cancer, wherein a T cell-mediated immune response is modulated or the growth of tumor cells is inhibited. delete delete delete delete

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